Cryopreservation of apoptotic cancer cells for use in immunotherapy against cancer

ABSTRACT

Described herein is a reliable method for preparing a potent vaccine useful for immunotherapy comprising the step of cryopreserving a population of cells undergoing immunogenic cell death, and using such cells to activate dendritic cells for use in immunotherapy. In a specific embodiment, the method comprises cryopreserving cancer cells undergoing cell death, which can be used to prepare a pharmaceutical composition for immunotherapy against cancer.

1. FIELD

Described herein is a method for generating pharmaceutical compositionsfor use in immunotherapy. In specific embodiments, methods forcryopreserving cancer cells undergoing immunogenic cell death areprovided for use in generating pharmaceutical compositions that can beused to activate dendritic cells for immunotherapy.

2. BACKGROUND 2.1 Dendritic Cell Immunotherapy

There is great interest in developing effective autologousimmunotherapeutic vaccines for treating or preventing human cancer.Success at such autologous immunotherapy requires the development of avaccine that is both specific for the patient's cancer and capable ofeliciting a potent immune response.

Autologous dendritic cell therapy is an approach that involves priming apatient's immune system to attack the patient's cancer cells. In thisprocedure, certain immune cells, namely dendritic cells (DCs), areexposed to, or “pulsed” with, cancer cells comprising the patient'sspecific cancer antigens. The dendritic cells pulsed with specificcancer antigens then present these antigens to naïve T lymphocytes ofthe immune system. This leads to the priming of the T lymphocytes into apopulation of effector T lymphocytes, which attack cancer cells in thepatient's body presenting those antigens.

To produce an autologous dendritic cell cancer vaccine, monocytes arecollected from a cancer patient by leukapheresis. The monocytes are thendifferentiated in vitro into DCs, which are then pulsed with cancercells related to the type of cancer being treated, and re-administratedto the patient to elicit an immune response against its cancer.

2.2 Cell Death

Cell death can be broadly classified into two types: necrosis andapoptosis. Necrosis is cell death caused by external factors damagingcells or tissue, whereas apoptosis is a programmed cell death whichoccurs naturally in living organisms. Cell death through either necrosisor apoptosis can be immunogenic or non-immunogenic. A specific type ofapoptosis, characterized as immunogenic cell death (ICD), has beenidentified as being capable of inducing a robust immunogenic response.(G. Kroemer et al., 2013, Immunogenic Cell Death in Cancer therapy. Ann.Rev. Immunol., vol 31, pp. 51-72). Cells undergoing ICD arepreferentially recognized by DCs, and can be used to pulse DCs, which,in turn, expose antigen from these dying cells to the immune system.

Despite the recent advances in the field of immunotherapy, the challengeof standardizing a process for producing an autologous cancer vaccinewhile ensuring optimal and reproducible antigen presentation by DCsafter pulsing with cancer cells remains unmet.

3. SUMMARY

In one aspect, provided herein are methods and compositions forgenerating highly potent and immunogenic vaccines. In one embodiment,described herein is a method for producing a population of animal cellsuseful for immunotherapy, comprising: (a) inducing ICD in a populationof animal cells expressing an antigen(s) of interest; (b) cryopreservingthe cells undergoing ICD; and (c) thawing the cryopreserved cells. Inanother embodiment, described herein is a method for producingantigen-pulsed dendritic cells, comprising (a) inducing immunogenic celldeath (ICD) in a population of animal cells expressing an antigen(s) ofinterest; (b) cryopreserving the cells undergoing ICD; (c) thawing thecells; and (d) pulsing dendritic cells with the thawed cells. In certainembodiments, the cells are expanded in cell culture prior to theinduction of ICD. In specific embodiments, the animal cells express acancer antigen(s) of interest. In some embodiments, the animal cells arecells derived from a subject having a disease. In a specific embodiment,the animal cells are from a cell line. In certain embodiments, theanimal cells are from one, two, three or more cell lines. In a specificembodiment, the animal cells are human cells.

In another embodiment, described herein is a method for preparing apharmaceutical composition for use in immunotherapy, comprising: (a)inducing ICD in a population of animal cells expressing an antigen(s) ofinterest; (b) cryopreserving the cells undergoing ICD; (c) thawing thecryopreserved cells; and (d) preparing a pharmaceutical compositioncomprising the thawed cells for use in immunotherapy. In certainembodiments, the cells are expanded in cell culture prior to theinduction of ICD. In another embodiment, described herein is a methodfor producing a composition for immunotherapy, comprising: (a) expandingcells from an animal cell line expressing antigens of interest; (b)inducing the cells to undergo ICD; (c) cryopreserving the expanded cellsundergoing ICD; (d) thawing the cryopreserved cells and (e) pulsing apopulation of dendritic cells with the thawed cells. In specificembodiments, the animal cells express cancer antigens of interest. Insome embodiments, the animal cells are cells derived from a subjecthaving a disease. In a specific embodiment, the animal cells are from acell line. In certain embodiments, the animal cells are from one, two,three or more cell lines. In a specific embodiment, the animal cells arehuman cells.

In another embodiment, provided herein is a batch of vials comprisingcryopreserved animal cells expressing an antigen(s) of interest. Inspecific embodiments, the animal cells express cancer antigens ofinterest. In a specific embodiment, the animal cells are from a cellline. In some embodiments, the animal cells are cells derived from asubject having a disease. In certain embodiments, the animal cells arefrom one, two, three or more cell lines. In a specific embodiment, theanimal cells are human cells. In a specific embodiment, the batch ofvials was produced by a method comprising: (a) inducing the cells toundergo ICD; and (b) cryopreserving the cells. In certain embodiments,the method further comprises expanding the cells prior to inducing thecells to undergo ICD. In some embodiments, a batch of vials is 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more vials.

In certain embodiments of the methods and compositions described herein,a single cell line is utilized. In other embodiments of the methods andcompositions described herein, a combination of multiple (e.g., 2, 3, 4,5, 6 or more) cell lines is utilized. In a specific embodiment, the cellline or cell line(s) selected for use in the methods and compositionsdescribed herein is based on the antigens expressed by the cell line orcell lines. In some embodiments, the cell line or cell lines selectedfor use in the methods and compositions described herein is according tothe different sub-types of cancer or other disease (e.g., infectiousdisease) to be treated with immunotherapy. In certain embodiments, thecell line or cell lines selected for use in the methods and compositionsis based on the antigens expressed by the cell line or cell lines andthe sub-type of the cancer or other disease (e.g., infectious disease)to be treated with immunotherapy.

In a specific aspect, provided herein are methods and compositions forgenerating highly potent and immunogenic cancer vaccines specific to apatient's tumor. This aspect is based, in part, on the discovery anddemonstration that cryopreservation of cancer cells in the criticalstage of immunogenic cell death (ICD) is capable of producing a potentand highly effective vaccine against human cancer.

Because cancer cell undergoing ICD can ensure an optimal maturation ofdendritic cells (DCs), activation of DCs by the dying cancer cells, andantigen presentation, cryopreservation of cancer cells at the stage ofundergoing ICD has many advantages over alternative methods of producingand maintaining cancer cells and cell lines, including: (1) the abilityto produce a standardized reagent that may be used to generatesufficient cancer vaccine for a series of immunotherapeutic treatmentsfor multiple patients; (2) yielding a product that can be fullycharacterized and quality-control tested before its use in amanufacturing process; (3) eliminating the possibility of phenotypicdrift in the tumor cell culture due to genetic instability or selectivepressure; (4) eliminating the cost required to maintain cell cultures;and (5) decreasing the risk of exposure to microbes and tocross-contamination. Thus, the process of cryopreserving large amountsof tumor cell lines at a highly immunogenic stage of cell death not onlystandardizes the manufacturing process, but produces a potent autologouscancer vaccine capable of eliciting a vigorous and specific immuneresponse in the cancer patient.

The use of cell lines to prepare the cancer vaccine provides thepossibility of producing a large amount of cancer cells expressing thesame cancer antigens, which, upon induction of ICD, can be cryopreservedin the form of ready-to-use aliquots of dying cancerous cells. Aftercryopreservation, each aliquot of the batch of cancer cells, uponthawing, retains the same physiological status, phenotype and genotype,immunogenicity and function properties as every other aliquot of thebatch. Thus, the cryopreservation of dying cells undergoing ICD allowsfor standardization of the process, because DCs from any patient can bepulsed with a phenotypically and genotypically identical cancer cellpopulation, expressing the same antigens in the same amounts, in whichcell death was induced at the same time and in the same way. Suchstandardization allows for quality control and optimization of thepreparation of cancer vaccines based on activated DCs.

In one embodiment, described herein is a method for producing apopulation of cancer cells useful for immunotherapy, comprising: (a)inducing ICD in a population of cancer cells; (b) cryopreserving thecancer cells undergoing ICD; and (c) thawing the cryopreserved cancercells. In another embodiment, described herein is a method for producingantigen-pulsed dendritic cells, comprising (a) inducing immunogenic celldeath (ICD) in a population of cancer cells; (b) cryopreserving thecancer cells undergoing ICD; (c) thawing the cancer cells; and (d)pulsing dendritic cells with the thawed cancer cells. In certainembodiments, the cancer cells are expanded in cell culture prior to theinduction of ICD. In some embodiments, the cancer cells are cellsderived from a subject having cancer. In a specific embodiment, thecancer cells are from a cancer cell line. In certain embodiments, thecancer cells are from one, two, three or more cancer cell lines. In aspecific embodiment, the cancer cells are human cancer cells.

In another embodiment, described herein is a method for preparing apharmaceutical composition for use in immunotherapy, comprising: (a)inducing ICD in a population of cancer cells; (b) cryopreserving thecancer cells undergoing ICD; (c) thawing the cryopreserved cancer cells;and (d) preparing a pharmaceutical composition comprising the thawedcancer cells for use in immunotherapy. In certain embodiments, thecancer cells are expanded in cell culture prior to the induction of ICD.In another embodiment, described herein is a method for producing acomposition for immunotherapy, comprising: (a) expanding cancer cells;(b) inducing the cancer cells to undergo ICD; (c) cryopreserving theexpanded cancer cells undergoing ICD; (d) thawing the cryopreservedcancer cells and (d) pulsing a population of dendritic cells with thethawed cancer cells. In some embodiments, the cancer cells are cellsderived from a subject having cancer. In a specific embodiment, thecancer cells are from a cell line. In certain embodiments, the cancercells are from one, two, three or more cancer cell lines. In a specificembodiment, the cancer cells are human cancer cells.

In another embodiment, provided herein is a batch of vials comprisingcryopreserved cancer cells. In a specific embodiment, the cancer cellsare from a cancer cell line. In some embodiments, the cancer cells arecells derived from a subject having cancer. In certain embodiments, thecancer cells are from one, two, three or more cancer cell lines. In aspecific embodiment, the cancer cells are human cancer cells. In aspecific embodiment, the batch of vials was produced by a methodcomprising: (a) inducing the cancer cells to undergo ICD; and (b)cryopreserving the cancer cells. In certain embodiments, the methodfurther comprises expanding the cancer cells prior to inducing the cellsto undergo ICD. In some embodiments, a batch of vials is 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more vials.

In another embodiment, described herein is a vial comprising apopulation cancer cells, wherein the population of cancer cells are acryopreserved population of cancer cells undergoing immunogenic celldeath.

In certain embodiments of the methods and compositions described herein,the cancer cell line is prostate cancer cell line, ovarian cancer cellline, or lung cancer cell line. In some embodiments of the methods andcompositions described herein, the cancer cell lines is a glioblastomacell line, renal cancer cell line, colon cancer cell line, or breastcancer cell line.

In certain embodiments of the methods and compositions described herein,a single cancer cell line is utilized. In other embodiments of themethods and compositions described herein, a combination of multiple(e.g., 2, 3, 4, 5, 6 or more) cancer cell lines is utilized. In aspecific embodiment, the cancer cell line or cancer cell line(s)selected for use in the methods and compositions described herein isbased on the antigens expressed by the cancer cell line or cancer celllines. In some embodiments, the cell line or cell lines selected for usein the methods and compositions described herein is according to thedifferent sub-types of cancer to be treated with immunotherapy. Incertain embodiments, the cancer cell line or cancer cell lines selectedfor use in the methods and compositions is based on the antigensexpressed by the cancer cell line or cancer cell lines and the sub-typeof the cancer to be treated with immunotherapy.

In another embodiment of the methods and compositions, population ofcancer cells used to pulse a population of dendritic cells is acancerous cell line. In another embodiment, a plurality of cancer celllines related to a same type of cancer is combined. In a specificembodiment, the cancer is a solid tumor cancer. In certain embodiments,the solid tumor cancer is prostate cancer, ovarian cancer, lung cancer,renal cancer, colon cancer, breast cancer or glioblastoma.

In one embodiment, the cancer to be treated by immunotherapy is prostatecancer and the cancerous cell line to be used to activate DCs is theLNCap cell line. In another embodiment, the cancer to be treated byimmunotherapy is ovarian cancer and two cancerous cell lines are used toactivate DCs, namely the cell line SK-OV-3 and the cell line OV-90. Inyet another embodiment, the cancer to be treated by immunotherapy islung cancer and two cells lines are used to activate DCs, namelyNCI-H520 and NCI-H522 cells lines.

In a specific embodiment, any pharmacological or mechanical inducer ofICD can be used to induce immunogenic cell death of the cells to becryopreserved. Identified inducers of ICD include: anthracyclines,anti-EGFR antibodies, Big Potassium channel antagonists, bortezomib,cardiac glycosides, cyclophosphamide, GADD43/PP1 inhibitors andmitomycin, irradiation by UV light or gamma rays, oxaliplatin,photodynamic therapy with hypericin, poly (I:C), thapsigargin andcisplatin, high hydrostatic pressure. In certain embodiments of themethods described herein, ICD is induced by high hydrostatic pressure,anthracyclines, anti-EGFR antibodies, Big Potassium channel antagonists,bortezomib, cardiac glycosides, cyclophosphamide, GADD43/PP1 inhibitorsand mitomycin, irradiation by UV light or gamma rays, oxaliplatin,photodynamic therapy with hypericin, poly(I:C), thapsigargin andcisplatin, or any other agent inducing immunogenic cell death. In apreferred embodiment, high hydrostatic pressure is used to induce ICD.In another embodiment, UV is used to induce ICD. In another embodiment,anthracyclines are used to induce ICD. In another embodiment,photodynamic therapy with hypericin is used to induce ICD. The regimenand type of pharmacological inducers of ICD might change from one cell(e.g., one cancerous cell) to another to obtain an optimal a maturationand activation of DCs.

In certain embodiments of the methods and compositions described herein,the animal cells (e.g., cancer cells) undergoing immunogenic cell deathare preserved in a cryopreservant solution. In a specific embodiment ofthe methods and compositions described herein, animal cells (e.g.,cancer cells) undergoing immunogenic cell death are preserved in acryopreservant solution containing at least 5% of dimethyl sulfoxide. Inanother embodiment of the methods and compositions described herein,animal cells (e.g., cancer cells) undergoing immunogenic cell death arepreserved in a cryopreservant solution containing at least 5% ofglycerol.

In another embodiment of the methods and compositions described herein,cells undergoing ICD are kept frozen at temperatures below −75° C. In aspecific embodiment of the methods and compositions described herein,cells undergoing ICD are kept frozen at temperatures of at least orbelow −130° C. In another embodiment, dying cells in cryopreservant arecooled at a rate of −1 to −5° C./min. In another embodiment of themethods and compositions described herein, dying cells are exposed totemperatures of −25 to −30° C. for up to 30 min before transferring tolower temperatures such as −130° C.

In another of the methods and compositions described herein, thealiquots of dying cells are transferred to a heavily insulated box andplaced at −80° C. for 24 hours and then transferred to lowertemperatures such as −130° C. In another embodiment of the methods andcompositions described herein, the aliquots of dying cells aretransferred to cooling boxes containing 100% isopropyl alcohol for 24hours, allowing freezing at a rate close to −1° C./min when place at−80° C. The aliquots can then transferred to lower temperatures such as−130° C.

In another embodiment, the dying cells are cryopreserved immediatelyafter induction of cell death by pharmacological or mechanical agents.In another embodiment of the methods and compositions described hereinthe dying cells are kept into culture for up to six hours after theinduction of immunogenic cell death prior to cryopreservation.

In another embodiment of the methods and compositions described herein,the thawed cells undergoing ICD are put back into culture in culturemedia for at least one hour and not more than six hours before beingincubated with DCs.

In an embodiment of the methods and compositions described herein, thehallmarks of immunogenic cell death are preserved duringcryopreservation. In another specific embodiment of the methods andcompositions described herein, after thawing, the cells undergoing celldeath retain the hallmarks of immunogenic cell death. In anotherembodiment of the methods and compositions described herein, the type ofcell death before and after freezing is the same type of cell death.

In another aspect, provided herein is a pharmaceutical pack or kitcomprising one or more containers comprising cryopreserved cellsundergoing ICD. The pharmaceutical pack or kit may include instructionsfor use of the cryopreserved cells described herein.

4. DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, and 1E. Cryopreservation of HHP-treated tumorcells preserves the apoptotic character of cell death and the expressionof immunogenic cell death markers. (A) The prostate cell line (LNCap)and ovarian cell line (SKOV3) were treated by HHP and cryopreservedafter time 0 and 4H. After thawing process in the time points OH and 4H,the apoptosis level as the percentage of early (annexin V+/DAPI−) andlate (annexinV+/DAPI+) apoptotic cells was determined by flow cytometry.(B) Dot plots of the representative experiments are shown. Cells werelabeled with DAPI and annexin V-Alexa 647. (C) The kinetics of HSP70,HSP90 and calreticulin surface expression by the prostate LNCap cellline and SKOV3 cell line following treatment with 250 MPa for 10 min andcryopreservation and thawing process. The expression of the indicatedmarkers by treated cells is shown as the mean fluorescence intensity(MFI). The compiled results of a total of 3 experiments are shown. *,P<0.05. (D) Stability of expression of IMM during the long time courseof 3 months for prostate (LNCap) and ovarian (SKOV3) cell line. (E)Confocal microscopy images of HHP-treated cells, cryopreserved andthawed in different time points.

FIGS. 2A and 2B. HHP treatment and cryopreservation of tumor cellsinduce positively an accumulation of tumor antigens compared to othercytoskeletal proteins. (A) Expression of PSA and PSMA tumor antigens onthe cell surface of prostate LNCap cancer cells and the expression ofHer2/Neu tumor antigen on the cell surface of SKOV3 cancer cell lineafter HHP treatment and cryopreservation and thawing process indifferent time points by flow cytometry. The MFI values of 3 independentexperiments are shown. *, P<0.05 as compared to untreated tumor cells.(B) The whole cell amount of PSA, PSMA and Her2/Neu proteins inHHP-treated cryopreserved cells by western blot analysis. The data showthe compiled results (mean±SD) of 3 independent experiments. *, P<0.05as compared to untreated tumor cells.

FIGS. 3A, 3B, and 3C. HHP-frozen tumor cells are phagocytosed by DCs atthe same level as non-frozen cells. (A) Killed LNCap and SKOV3 celllines were labeled with DiO and cocultured with DiD-labeled immatureDCs. Percentage of phagocytosis at 24 h following HHP treatment andcryopreservation as compared to control. (B) Dot plots of representativeexperiments are shown. (C) Immature DCs (day 5) were cultured for 24 hwith LNCap cells and SKOV3 cells treated by HHP and cryopreserved. After24 h, the expression of CD80, CD83, CD86 and CCR7 by DCs was analyzed byflow cytometry. The MFI values are shown of 3 independent experiments.*, P<0.05. as compared to untreated tumor cells.

FIGS. 4A and 4B. DCs pulsed with HHP-frozen tumor cells inducetumor-specific T cells. (A) Monocyte-derived DCs were pulsed with LNCapor SKOV3 cells killed by HHP and cryopreserved and then used tostimulate autologous T cells for 2 weeks. The number of IFN-γ-producingcells in cultures with unpulsed DCs or DCs pulsed with tumor cellsnon-frozen and frozen was analyzed by intracellular IFN-γ staining afterrestimulation. The data show a summary and (B) representative stainingof 3 independent experiments. *, P<0.05 as compared to untreated tumorcells.

5. DETAILED DESCRIPTION 5.1 Terminology

In describing and claiming the invention, the following terms should beunderstood as follows.

The term “isolated cell” or “isolated population of cells” means anycell or population of cells of any organism that does not naturallyoccur in nature. The term “isolated host cell” means any cell of anyorganism that is removed from its natural environment and selected,modified, transformed, grown, used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Isolatedcells, population of cells, host cells, and genetically engineered cellsinclude isolated immune cells or a population of immune cells, such asmonocytes and dendritic cells.

To “expand,” “specifically expand” or “preferentially expand” in thecontext of dendritic cells means to culture a population of monocytes ina media containing growth factor inducing the proliferation anddifferentiation of the monocytes into immature dendritic cells. In apreferred embodiment, monocytes are expended and differentiated into DCswith GM-CSF and IL4.

An “autologous cell” refers to a cell which was derived from the sameindividual that is being treated by cell therapy.

A “donor cell” refers to a cell that was derived from an individualother than the individual being treated by cell therapy.

An “allogeneic cell” refers to a genetically distinct cell.

As used herein, the terms “treat,” “treating,” and “treatment” in thecontext of the administration of a therapy to a subject refer to thebeneficial effects that a subject derives from a therapy. In certainembodiments, treatment of a subject with a cancer vaccine describedherein achieves at least one, two, three, four or more of the followingeffects: (i) the reduction or amelioration of the severity of one ormore symptoms of cancer; (ii) the reduction in the duration of one ormore symptoms associated with cancer; (iii) the protection against therecurrence of a symptom associated with cancer; (iv) the reduction inhospitalization of a subject; (v) a reduction in hospitalization length;(vi) the increase in the survival of a subject; (vii) the enhancement orimprovement of the therapeutic effect of another therapy; (viii) anincrease in the survival rate of patients; (xiii) a decrease inhospitalization rate; (ix) the protection against the development oronset of one or more symptoms associated with cancer; (x) the reductionin the number of symptoms associated with cancer; (xi) an increase insymptom-free survival of cancer patients; (xii) improvement in qualityof life as assessed by methods well known in the art; (xiii) theprotection against the recurrence of a tumor; (xiv) the regression oftumors and/or one or more symptoms associated therewith; (xvii) theinhibition of the progression of tumors and/or one or more symptomsassociated therewith; (xviii) a reduction in the growth of a tumor;(xix) a decrease in tumor size (e.g., volume or diameter); (xx) areduction in the formation of a newly formed tumor; (xxi) eradication,removal, or control of primary, regional and/or metastatic tumors;(xxii) a decrease in the number or size of metastases; (xxiii) areduction in mortality; (xxiv) an increase in the tumor-free survivalrate of patients; (xxv) an increase in relapse free survival; (xxvi) anincrease in the number of patients in remission; (xxvii) the size of thetumor is maintained and does not increase or increases by less than theincrease of a tumor after administration of a standard therapy asmeasured by conventional methods available to one of skill in the art,such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI(DCE-MRI), X-ray, and computed tomography (CT) scan, or a positronemission tomography (PET) scan; r (xxviii) an increase in the length ofremission in patients; and/or (xxiv) a decrease in measurable cancerantigens

As used herein, term “protecting against” in the context ofadministering a therapy to a subject refers to the prophylactic effectthat a subject receives from a therapy. In a specific embodiment, thisterm refers to the inhibition of the development or onset of cancer or asymptom associated therewith, or inhibition of the recurrence of canceror a symptom thereof.

As used herein, the terms “manage,” “managing,” and “management,” in thecontext of the administration of a therapy to a subject, refer to thebeneficial effects that a subject derives from a therapy, which does notresult in a cure of a disease. In certain embodiments, a subject isadministered one or more therapies to “manage” cancer so as to preventthe progression or worsening of symptoms associated with the cancer.

As used herein, the terms “subject” and “patient” are usedinterchangeably and refer to an animal. In a specific embodiment, suchterms refer to a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), mostpreferably a human.

As used herein, the term “significant,” as in “significant” amount,change or effect, for example, means that the amount, change, or effectproduced would not be likely to have occurred by random chance, asdetermined by any standard method for statistical analysis, such as a ptest, wherein a p value less than the critical alpha level indicatesthat an event would be unlikely. Thus, a “significant” change in thecontext of this invention indicates the p value is less than thecritical alpha level, and that the probability is small that the changehappened by chance.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.Examples of effective amounts are provided in Section 5.9.2, infra.

As used herein “not significantly altered” means a change in that aamount or effect produced would is not statistically significant, asdetermined by any standard method for statistical analysis, such as a ptest. In a specific embodiment, the p value is 0.5, 0.1, 0.05, 0.001,0.005, or 0.0001.

All terms used herein, unless otherwise defined, will be given theirordinary technical or scientific meaning as would be commonly understoodby one of ordinary skill in the art at the time of the disclosure.

5.2 Methods for Inducing Immunogenic Cell Death

In one aspect, provided are methods for inducing a specific type ofapoptotsis characterized as immunogenic cell death (“ICD”) in animalcells expressing an antigen(s) of interest. In a specific aspect,provided are methods for inducing a specific type of apoptotsischaracterized as immunogenic cell death (“ICD”) in cancer cells. ICD isthe only type of cell death which is capable of producing a robustimmune response against antigens expressed by dying cells. The hallmarksof ICD are the expression of immunogenic molecules on the cell surfacesuch as HSP70, HSP90 and calreticulin and the release of late apoptoticmarkers HMGB1 and ATP. The interaction of DCs with a cell, such as acancer cell, dying under ICD conditions leads to a more rapid rate of DCphagocytosis and significant maturation of DCs. DCs treated with ICDdying cancer cells induce high numbers of tumor-specific T lymphocytes.

Any technique known in the art may be used to induce ICD of animal cellsexpressing an antigen(s) of interest (e.g., cancer cells). As a personskilled in the art will appreciate, not all techniques known to induceapoptosis will necessarily induce ICD. Only apoptotic agents inducingICD will elicit the most efficient DC maturation.

In one embodiment, a pharmacological agent(s) is used to induce ICD. Inanother embodiment, a mechanical technique(s) is used to induce ICD. Inanother embodiment, a combination of a pharmacological agent(s) and amechanical technique(s) is used to induce ICD. The regimen and type ofpharmacological inducer(s) and/or mechanical inducer(s) of ICD mightvary depending on the cancerous cells. The pharmacological inducersand/or mechanical inducers of ICD used to obtain an optimal maturationand activation of DCs may differ from one type of cancer cell to anothertype of cancer cell.

Examples of inducers of ICD include, but are not limited to,anthracyclines, anti-EGFR antibodies, Big Potassium channel antagonists,bortezomib, cardiac glycosides, cyclophosphamide, GADD43/PP1 inhibitorsand mitomycin, irradiation by UV light or gamma rays, oxaliplatin,photodynamic therapy with hypericin, poly (I:C), thapsigargin andcisplatin, and high hydrostatic pressure. In a specific embodiment, highhydrostatic pressure (“HHP”) is used to induced ICD. See, e.g.,International Publication No. WO 2013/004708 A1 for a discussion of ICDinduced by HHP. In another embodiment, UV light is used to induce ICD.In another embodiment, anthracyclines are used to induce ICD. In anotherembodiment, photodynamic therapy with hypericin is used to induce ICD.

The hallmarks of ICD are the expression of immunogenic molecules on thecell surface, such as HSP70, HSP90 and calreticulin, and the release oflate apoptotic markers HMGB1 and ATP. Techniques known to one skilled inthe art can be used to assess the expression of these cell surfacemarkers and the release of the late apoptotic makers. For example, theexpression of the cell surface markers can be assessed using standardtechniques such as flow cytometry, immunocytochemistry (e.g., stainingwith tissue specific or cell-marker specific antibodies), fluorescenceactivated cell sorting (FACS), and magnetic activated cell sorting(MACS). Fluorescence activated cell sorting (FACS) is a well-knownmethod for separating particles, including cells, based on thefluorescent properties of the particles (Kamarch, 1987, Methods Enzymol,151:150-165). Laser excitation of fluorescent moieties in the individualparticles results in a small electrical charge allowing electromagneticseparation of positive and negative particles from a mixture. In oneembodiment, cell surface marker-specific antibodies or ligands arelabeled with distinct fluorescent labels. Cells are processed throughthe cell sorter, allowing separation of cells based on their ability tobind to the antibodies used. FACS sorted particles may be directlydeposited into individual wells of 96-well or 384-well plates tofacilitate separation and cloning. The release of the HMGB1 can beassessed by, e.g., enzyme-linked immunosorbent assay-based methodologies(ELISA), western blot, or other similar methods known in the art.

Apoptosis can be quantitated by measuring DNA fragmentation. Commercialphotometric methods for the quantitative in vitro determination of DNAfragmentation are available. Examples of such assays, including TUNEL(which detects incorporation of labeled nucleotides in fragmented DNA)and ELISA-based assays, are described in Biochemica, 1999, no. 2, pp. 3437 (Roche Molecular Biochemicals). Apoptosis can also be observedmorphologically. Apoptosis can also be assessed by annexin V fluoresceinstaining, such as described in Section 6, infra.

In a specific embodiment, one or more of the assays described in Section6, infra, can be utilized to assess the induction of ICD.

In specific embodiments, the cancer cells induced to undergo ICD may becryopreserved immediately, or a few minutes (e.g., 10 minutes, 15minutes, 30 minutes, or 45 hours) or a few hours (e.g., within 1 hour,1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, or 8 hours)after induction of ICD. In one embodiment, cancer cells induced toundergo ICD are kept in cell culture for at least one hour and not morethan six hours before being cryopreserved. In a specific embodiment,cancer cells induced to undergo ICD kept in cell culture for 0.5 hoursto 1 hour, 1 hour to 2 hours, 1.5 hours to 2 hours, 1.5 hours to 2.5hours, 2 hours to 3 hours, 2 to 4 hours, 3 hours to 4 hours, 4 hours to5 hours, 3 hours to 6 hours, 4 hours to 6 hours, 5 hours to 6 hours, 2to 4 hours, 2 to 5 hours, 3 to 5 hours, 2 hours to 6 hours, or 3 hoursto 6 hours before being cryopreserved. In another specific embodiment,cancer cells induced to undergo ICD kept in cell culture for 1.5 hours,2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5hours or 6 hours before being cryopreserved.

In a certain embodiments, the cancer cells induced to undergo ICD arecharacterized prior to cryopreservation using techniques describedherein (e.g., in this Section 5 or Section 6, infra) or known to oneskilled in the art for characterizing cells undergoing ICD. In aspecific embodiment, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% of the cancer cells induced to undergoICD are characterized as undergoing ICD using techniques describedherein or known to one skilled in the art before cryopreservation. Inanother embodiment, 50% to 75%, 50% to 99%, 75% to 85%, 75% to 90%, 75%to 99%, 80% to 90%, 80% to 99%, 85% to 90%, 85% to 95%, or 95% to 99% ofthe cancer cells induced to undergo ICD are characterized as undergoingICD using techniques described herein or known to one skilled in the artbefore cryopreservation.

In another specific embodiment, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98% of the cancer cellsinduced to undergo ICD are characterized as undergoing ICD by theexpression of one or more ICD markers, such as HSP70, HSP90, orcalreticulin, using techniques described herein or known to one skilledin the art before cryopreservation. In another embodiment, 50% to 75%,50% to 99%, 75% to 85%, 75% to 90%, 75% to 99%, 80% to 90%, 80% to 99%,85% to 90%, 85% to 95%, or 95% to 99% of the cancer cells induced toundergo ICD are characterized as undergoing ICD by the expression of oneor more ICD markers, such as HSP70, HSP90, or calreticulin, usingtechniques described herein or known to one skilled in the art beforecryopreservation.

5.3 Type of Cancer Cells Induced to Undergo Immunogenic Cell Death

In one aspect, the cancer cells induced to undergo ICD are from a cancercell line. The type of cancer cell line can be chosen accordingly to thetype of cancer to be treated in the patient or to the type of antigensexpressed by the cancer cell line. Multiple cancer cell lines related toa same type of cancer can be combined to assure a broad spectrum ofexpressed antigens and a more efficient cancer vaccine.

In a specific embodiment, the cancer cells induced to undergo ICD arefrom a solid tumor. In certain embodiments, the cancer cells induced toundergo ICD are mesothelioma cells, melanoma cells, adenoma cells,carcinoma cells, adenocarcinoma cells, ductal carcinoma cells,rhabdomyosarcoma cells, osteosarcoma cells, neuroblastoma cells,astrocytoma cells, or glioblastoma cells. In one embodiment, the cancercells induced to undergo ICD are from a carcinoma, such as anadenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, acolorectal adenocarcinoma, a colorectal carcinoma, a ductal cellcarcinoma, a lung carcinoma, a thyroid carcinoma, a hepatocellularcarcinoma, a nasopharyngeal carcinoma, or an unspecified carcinoma. Inanother embodiment, the cancer cells induced to undergo ICD are from amelanoma (e.g., a malignant melanoma), a non-melanoma skin carcinoma. Inanother embodiment, the cancer cells induced to undergo cell deathICDare from a desmoid tumor, a desmoplastic small round cell tumor; anendocrine tumor, an Ewing sarcoma, a germ cell tumor (e.g., testicularcancer, ovarian cancer, choriocarcinoma, endodermal sinus tumor,germinoma, etc.), a hepatosblastoma, a neuroblastoma; anon-rhabdomyosarcoma soft tissue sarcoma; an osteosarcoma, aretinoblastoma, a rhabdomyosarcoma, or a Wilms tumor. In anotherembodiment, the cancer cells induced to undergo ICD are from an acousticneuroma; an astrocytoma (e.g., a grade I pilocytic astrocytoma, a gradeII low-grade astrocytoma, a grade III anaplastic astrocytoma, or a gradeIV glioblastoma multiforme); a chordoma; a craniopharyngioma; a glioma(e.g., a brain stem glioma; an ependymoma; a mixed glioma; an opticnerve glioma; or a subependymoma); a glioblastoma; a medulloblastoma; ameningioma; a metastatic brain tumor; an oligodendroglioma; apineoblastoma; a pituitary tumor; a primitive neuroectodermal tumor; ora schwannoma. In another embodiment, the cancer cells induced to undergoICD are from pancreatic cancer, breast cancer, brain cancer, renalcancer, prostate, cervical cancer, liver cancer, colorectal cancer,ovarian cancer, colon cancer, testicular cancer, thyroid cancer, lungcancer, or breast cancer. In a certain embodiments, the cancer cellsinduced to undergo ICD are from a malignant tumor. In a specific aspect,in accordance with the embodiments in this paragraph, the cancer cellsfrom these types of cancers/tumors are cell lines.

In another embodiment, the cancer cells induced to undergo ICD are froma benign tumor. In another embodiment, the cancer cells induced toundergo ICD are from a solid tumor cancer comprising prostate cancer,ovarian cancer, lung cancer, renal cancer, colon cancer, breast canceror glioblastoma. In a specific aspect, in accordance with theembodiments in this paragraph, the cancer cells from these types ofcancers/tumors are cell lines.

In some embodiments, the cancer cells induced to undergo ICD areleukemia cells, acute myelogenous leukemia cells, acute myeloid leukemiacells, acute T cell leukemia cells, acute lymphoblastic leukemia cells,hairy cell leukemia cells, acute promyelocytic leukemia cells, lymphomacells, Burkitt's lymphoma cells, B cell chronic lymphocytic leukemiacells, non-Hodgkin's lymphoma cells, Hodgkin's lymphoma cells, ormultiple myeloma cells. In certain embodiments, the cancer cells inducedto undergo ICD are tumor stem cells or cancer stem cells. In a specificaspect, in accordance with the embodiments in this paragraph, the cancercells from these types of cancers are cell lines.

In certain embodiments, the cancer cells induced to undergo ICD arecancer cell line cells. Examples of cancer cell lines include, but arenot limited to, 5637 (Carcinoma), KHOS/NP (Osteosarcoma), MNNG/HOS(Osteosarcoma), Saos-2 (Osteosarcoma), U-2 OS (Osteosarcoma), SJSA-1(Osteosarcoma), CCF-STTG1 (Astrocytoma), DBTRG-05MG (Glioblastoma), U87MG (Glioblastoma), T98G (Glioblastoma), SK-N-SH (Neuroblastoma), SK-N-AS(Neuroblastoma), MCF-7 (Adenocarcinoma), MDA-MB-231 (Adenocarcinoma),MDA-MB-436 (Adenocarcinoma), SK-BR-3 (Adenocarcinoma), BT-20(Carcinoma), BT-474 (Carcinoma), CAMA-1 (Carcinoma), HCC2218(Carcinoma), SW527 (Carcinoma), MDA-MB-453 (Carcinoma), MDA-MB-4355(Carcinoma), T-47D (Carcinoma), ZR-75-1 (Carcinoma), UACC-812(Carcinoma), HCC1419 (Carcinoma), HeLa (Adenocarcinoma), Caco-2(Adenocarcinoma), COL0205 (Adenocarcinoma), COL0320/DM (Adenocarcinoma),DLD-1 (Adenocarcinoma), HCT-15 (Adenocarcinoma), SK-CO-1(Adenocarcinoma), SW48 (Adenocarcinoma), SW480 (Adenocarcinoma), HCT-8(Adenocarcinoma), RKO (Carcinoma), LS411N (Carcinoma), T84 (Carcinoma),AGS (Adenocarcinoma), KATO III (Carcinoma), NCI-N87 (Carcinoma), SNU-16(Carcinoma), 769-P (Adenocarcinoma), 786-O (Adenocarcinoma), ACHN(Adenocarcinoma), A-498 (Carcinoma), Caki-1 (Carcinoma), G-402(Leiomyoblastoma), CML-T1 (Leukemia), CTV-1 (Leukemia), JVM-2(Leukemia), K562 (Leukemia), MHH-CALL2 (Leukemia), NALM-6 (Leukemia),8E5 (Leukemia), CCRF-SB (Leukemia), CEM/C1 (Leukemia), CEM/C2(Leukemia), CEM-CM3 (Leukemia), CCRF-HSB-2 (Leukemia), KG-1 (Leukemia),KG-la (Leukemia), CCRF-CEM (Leukemia), MOLT-3 (Leukemia), SUP-B15(Leukemia), TALL-104 (Leukemia), Loucy (Leukemia), RS4;11 (Leukemia),REH (Leukemia), AML-193 (Leukemia), THP-1 (Leukemia), MOLM-13(Leukemia), Kasumi-1 (Leukemia), Kasumi-3 (Leukemia), BDCM (Leukemia),HL-60 (Leukemia), I 2.1 (Leukemia), I 9.2 (Leukemia), J.gammal.WT(Leukemia), J.RT3-T3.5 (Leukemia), P116 (Leukemia), P116.c139 [P116.c39](Leukemia), D1.1 (Leukemia), J45.01 (Leukemia), MV-4-11 (Leukemia),Kasumi-4 (Leukemia), MEG-01 (Leukemia), KU812 (Leukemia), Mo (Leukemia),JM1 (Leukemia), GDM-1 (Leukemia), CESS (Leukemia), ARH-77 (Leukemia),SK-HEP-1 (Adenocarcinoma), Bel-7402 (Carcinoma), Bel-7404 (Carcinoma),HEP-3B (Carcinoma), HepG2 (Carcinoma), Calu-3 (Adenocarcinoma),NCI-H1395 (Adenocarcinoma), NCI-H1975 (Adenocarcinoma), SK-LU-1(Adenocarcinoma), NCI-H2122 (Adenocarcinoma), NCI-H727 (Carcinoid),A-427 (Carcinoma), A549 (Carcinoma), SW1573 (Carcinoma), NCI-H358(Carcinoma), NCI-H460 (Carcinoma), NCI-H292 (Carcinoma), NCI-H82(Carcinoma), NCI-H226 (Carcinoma), NCI-H526 (Carcinoma), or MSTO-211H(Mesothelioma). In certain embodiments, the cancer cells induced toundergo ICD are from the ovarian cell line SK-OV3 or OV-90. In someembodiments, the cancer cells induced to undergo ICD are from the lungcancer cell line NCI-H520 or NCI-H522. In certain embodiments, thecancer cells induced to undergo ICD are from the prostate cancer cellline LNCap.

In certain embodiments, the cancer cells induced to undergo ICD are froma single cancer cell line. In other embodiments, the cancer cellsinduced to undergo ICD are from a combination of two, three, four, fiveor more cancer cell lines. For example, the cancer cells induced toundergo ICD may be from the SK-OV3 and OV-90 cell lines. In anotherexample, the cancer cells induced to undergo ICD may be from theNCI-H520 and NCI-H522 cell lines. In specific embodiments, the cancercells induced to undergo ICD are from a combination of cancer cell linesfrom the same type of cancer. For example, the cancer cells induced toundergo ICD may be from two, three, four, five or more lung cancer celllines. In another example, the cancer cells induced to undergo ICD maybe from two, three, four, five or more prostate cancer cell lines. Incertain embodiments, the cancer cells induced to undergo ICD are chosenbased upon the cancer antigens the cells express.

In some embodiments, the cancer cells induced to undergo ICD areobtained from a patient's tumor. Techniques known to one of skill in theart may be used to obtain cancer cells from a patient's tumor. In someembodiments, a biopsy of a patient's tumor is obtained. The biopsy canbe from any organ or tissue, for example, skin, liver, lung, heart,colon, kidney, bone marrow, teeth, lymph node, hair, spleen, brain,breast, or other organs. Any biopsy technique known by those skilled inthe art can be used for isolating a tumor sample from a patient, forinstance, open biopsy, close biopsy, core biopsy, incisional biopsy,excisional biopsy, or fine needle aspiration biopsy. In certainembodiments, the tumor biopsy is stored before the cancer cells areisolated. The tumor biopsy may be stored for 30 minutes, 60 minutes, 1hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours48 hours, 72 hours, 1 week, two weeks, three weeks, 1 month, 2 months ormore before cancer cells are isolated and induced to undergo ICD. Incertain embodiments, cancer cells isolated from a tumor biopsy arestored for a period of time before they are induced to undergo ICD. Thecancer cells may be stored for 15 minutes, 30 minutes, 60 minutes, 1hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours48 hours, 72 hours, 1 week, two weeks, three weeks, 1 month, 2 months ormore before they are induced to undergo ICD. In some embodiments, cancercells obtained from a tumor biopsy are immediately induced to undergoICD. In a specific embodiment, cancer cells obtained from a patient'stumor are utilized to create a cancer cell line and the cancer cell lineis induced to undergo ICD.

5.4 Methods for Cryopreserving Cells Induced to Undergo Immunogenic CellDeath

In one aspect, described herein is the use of cryopreservation, thelong-term storage of biological materials at very low temperature, tomaintain a reproducible source of cancer cells undergoing ICD forextended periods of time. In a specific aspect, the cancer cells are inthe process of dying under ICD conditions prior to cryopreservation.Cells prepared in this way retain the ICD conditions aftercryopreservation when thawed.

Methods for cryopreservation of cells are well known in the art. See,e.g., C. B. Morris, “Cryopreservaton of Animal and Human Cell Lines”(2007), in Methods in Molecular Biology, vol 368: Cryopreservation andFreeze-Drying Protocols, 2nd Ed. (J. G. Day and G. N. Stacey eds.),Humana Press Inc. Totowa, N.J., pp. 227-236, which is incorporatedherein in its entirety.

In one aspect, cryopreservant solutions are used to hold the cancercells in a fixed state of ICD for extended periods of time. The samecryopreservants used to maintain cells alive may be used to maintaindying cells is their specific ICD conditions during and aftercryopreservation.

Any cryoperservant available in the art may be used. In a specificembodiment, cancer cells undergoing ICD are preserved in acryopreservant solution containing at least 5% of dimethyl sulfoxide. Incertain embodiments, cancer cells undergoing ICD are preserved in acryopreservant solution containing 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% dimethyl sulfoxide. In someembodiments, cancer cells undergoing ICD are preserved in acryopreservant solution containing 5% to 10%, 10% to 15% or 15% to 20%dimethyl sulfoxide. In another embodiment, cancer cells undergoing ICDare preserved in a cryopreservant solution containing at least 5% ofglycerol. In certain embodiments, cancer cells undergoing ICD arepreserved in a cryopreservant solution containing 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of glycerol. Insome embodiments, cancer cells undergoing ICD are preserved in acryopreservant solution containing 5% to 10%, 10% to 15% or 15% to 20%glycerol.

Cryoperservants are commercially available; in one preferred embodimenta cryoperservant having 10% DMSO is used, such as the commerciallyavailable CryoStor CS10.

In one embodiment, to retain the cancer cells in the best conditionsduring cryopreservation, cancer cells are cooled at a constant slowrate. In one embodiment, cancer cells undergoing ICD in cryopreservantare cooled at a rate of −1 to −5° C./min, or −2 to −5° C./min, −3 to −5°C./min, −4 to −5° C./min, −5 to −6° C./min, −5 to −7° C./min, −5 to −8°C./min, −5 to −9° C./min, −5 to −10° C./min, −7 to −10° C./min, or −8 to−10° C./min. In another embodiment, cancer cells undergoing ICD incryopreservant are cooled at a rate of −1° C./min, −2° C./min, −3°C./min, −4° C./min, −5° C./min, −6° C./min, −7° C./min, −8° C./min, −9°C./min or −10° C./min. In another embodiment, cancer cells undergoingICD are exposed to temperatures of −25 to −30° C. or −30 to −35° C. forup to 30 min before transferring to lower temperatures such as −130° C.In another embodiment, cancer cells undergoing ICD are exposed totemperatures of −24° C., −25° C., −26° C., −27° C., −28° C., −29° C.,−30° C., −31° C., −32° C., −33° C., −34° C. or −35° C. to −30° C. for 1to 45 minutes (e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35minutes, 40 minutes or 45 minutes) before transferring to lowertemperatures such as −120° C., −125° C., −130° C., −135° C., or −140° C.

In one embodiment, to insure optimal cryopreservation, cancer cells incryopreservants are frozen and kept at very low temperatures. In oneembodiment, cancer cells undergoing ICD are kept frozen at temperaturesbelow −75° C. (e.g., −80° C., −85° C., −90° C., −95° C., −100° C., −105°C., −110° C., −115° C., −120° C., −125° C. or −130° C.). In a specificembodiment, cells undergoing ICD are kept frozen at temperatures of atleast or below −130° C. (e.g., −135° C., −140° C., −145° C., −150° C.,−155° C., −160° C., −165° C., −170° C., −175° C., or −180° C.).

In another embodiment, aliquots of cancer cells undergoing ICD aretransferred to a heavily insulated box and placed at −80° C. for 24hours and then transferred to lower temperatures such as −130° C., −135°C., −140° C., −145° C., −150° C., −155° C., −160° C., −165° C., −170°C., −175° C., or −180° C. In another embodiment, aliquots of cancercells undergoing ICD are transferred to cooling boxes containing 100%isopropyl alcohol for 24 hours, allowing freezing at a rate close to −1°C./min when place at −80° C. The aliquots are then transferred to lowertemperatures such as −130° C., −135° C., −140° C., −145° C., −150° C.,−155° C., −160° C., −165° C., −170° C., −175° C., or −180° C.

In a specific embodiment, cells undergoing ICD are cryopreserved asdescribed in Section 6, infra.

In a specific embodiment, aliquots of cancer cells undergoing ICD arecryopreserved as described herein. For examples, cancer cells undergoingICD may be aliquoted into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more containers, such as vials, and thencryopreserved as described herein. In another specific embodiment,cancer cells induced to undergo ICD may be cryopreserved incryopreservant solution as described herein and aliquoted in 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morecontainers, such as vials. In certain embodiments, a container (e.g., avial) of cryopreserved cancer cells contains 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000cancer cells induced to undergo ICD. In some embodiments, a container(e.g., a vial) of cryopreserved cancer cells contains 1×10², 5×10²,1×10³, 5×10³, 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷ or1×10⁸ cancer cells induced to undergo ICD. In certain embodiments, acontainer (e.g., a vial) of cryopreserved cancer cells contains 1×10¹ to1×10², 1×10² to 5×10², 1×10² to 1×10³, 1×10³ to 5×10³, 1×10² to 1×10⁴,1×10³ to 5×10⁴, 1×10³ to 1×10⁴, 1×10² to 1×10⁵, 1×10² to 5×10⁵, 1×10² to1×10⁶, 1×10³ to 5×10⁶, 1×10⁴ to 1×10⁷, 1×10⁵ to 1×10⁷, 1×10⁵ to 1×10⁷,1×10⁶ to 1×10⁸, 1×10⁷ to 1×10⁸ cancer cells induced to undergo ICD.

In some embodiments, a batch of cancer cells are induced to undergo ICD,aliquots of the cancer cells are cryopreserved in separate containers(e.g., 1 to 25, 25 to 50, 50 to 75, 75 to 100, 100 to 125, 125 to 150,150 to 200 or more containers, such as vials), and 1, 2 or 3 more of thevials may be thawed each time dendritic cells need to be pulsed withcancer cells to produce a cancer vaccine. In certain embodiments, acontainer (e.g, a vial) of cryopreserved cancer cells contains 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950 or 1000 cancer cells induced to undergo ICD. In someembodiments, a container (e.g., a vial) of cryopreserved cancer cellscontains 1×10², 5×10², 1×10³, 5×10³, 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶,5×10⁶, 1×10⁷, 5×10⁷ or 1×10⁸ cancer cells induced to undergo ICD. Incertain embodiments, a container (e.g., a vial) of cryopreserved cancercells contains 1×10¹ to 1×10², 1×10² to 5×10², 1×10² to 1×10³, 1×10³ to5×10³, 1×10² to 1×10⁴, 1×10³ to 5×10⁴, 1×10³ to 1×10⁴, 1×10² to 1×10⁵,1×10² to 5×10⁵, 1×10² to 1×10⁶, 1×10³ to 5×10⁶, 1×10⁴ to 1×10⁷, 1×10⁵ to1×10⁷, 1×10⁵ to 1×10⁷, 1×10⁶ to 1×10⁸, 1×10⁷ to 1×10⁸ cancer cellsinduced to undergo ICD.

5.5 Methods for Thawing Cryopreserved Cells

As for living cells, the way dying cells are recovered aftercryopreservation is crucial to maintain the preferred type of celldeath. In a preferred embodiment, cryopreserved cancer cells undergoingICD are thawed rapidly, preferably between 10 seconds and 5 mins. In oneembodiment, cryopreserved cells are thawed at a temperature of 32° C.,33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C. forbetween 5 secs. and 10 mins. In a specific embodiment, cells undergoingICD are thawed by warming a container (e.g., a vial) of cryopreservedcells in the palms of the hand for between 10 secs. and 5 mins. In aspecific embodiment, cryopreserved cancer cells undergoing ICD arethawed as described in Section 6, infra.

It is known that freezing and thawing, when non-optimal conditions areused, can induce cell death. Such cell death is typically necrosis,which is an undesired type of cell death for the maturation of DCs. Itis therefore critical when freezing and thawing dying cells to ensurethat ICD is maintained before and after cryopreservation.

In one embodiment, the cryopreserved cancer cells undergoing ICD arethawed and may be used immediately or a few hours after thawing for theincubation with DCs. In specific embodiments, the cells undergoing ICDmay be used immediately, or a few minutes (e.g., 10 minutes, 15 minutes,30 minutes, or 45 hours) or a few hours (e.g., within 1 hour, 1.5 hours,2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours) after thawing forthe incubation with DCs. In one embodiment, the thawed cells undergoingICD are put back into culture in culture media for at least one hour andnot more than six hours before being incubated with DCs. In a specificembodiment, the thawed cells undergoing ICD are put back into culture inculture media for 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours or 7 hoursbefore being incubated with DCs. In another specific embodiment, thethawed cells undergoing ICD are put back into culture in culture mediafor 0.5 to 1 hour, 1 to 2 hours, 1.5 hours to 2 hours, 2 to 3 hours, 2to 4 hours, 2.5 hours to 3 hours, 3 to 3.5 hours, 2 to 4 hours, 3 to 4hours, 4 to 4.5 hours, 4 to 5 hours, 4 to 6 hours, 5 to 6 hours, 1 to 6hours, 2 to 6 hours, or 5 to 7 hours before being incubated with DCs.

In a specific embodiment, cryopreserved cancer cells are thawed andcharacterized as undergoing ICD using techniques described herein (e.g.,in Section 5.2, supra, or Section 6, infra) or known to one skilled theart. In another specific embodiment, the cryopreserved cancer cellsinduced to undergo ICD are stable as assessed by the expression ofcertain markers, such as ICD markers (e.g., HSP70, HSP90 andcalreticulin). Techniques known to one skilled in the art, such asimmunofluorescence, can be used to assess the expression of suchmarkers. In a specific embodiment, the expression of ICD markers isassessed as described in Section 6, infra. In certain embodiments,greater than 50% of the cryopreserved cancer cells express ICD markersafter thawing. In specific embodiments, approximately 55%, approximately60%, approximately 65%, approximately 70%, approximately 75%,approximately 80%, approximately 85%, approximately 90%, approximately95% or approximately 98% of the cryopreserved cancer cells express ICDmarkers after thawing. In some embodiments, approximately 55% toapproximately 65%, approximately 65% to approximately 85%, orapproximately 75%, to approximately 85%, approximately 80% toapproximately 90% or approximately 90% to approximately 95% of thecryopreserved cancer cells express ICD markers after thawing. In aspecific embodiment, the expression of ICD markers, such as HSP70, HSP90and calreticulin, by cryopreserved cancer cells undergoing ICD afterthawing is not significantly altered relative tonon-cryopreserved/thawed cancer cells induced to undergo ICD.

In another embodiment, the expression of tumor antigens by cryopreservedcancer cells undergoing ICD after thawing is not significantly alteredrelative to non-cryopreserved/thawed cancer cells induced to undergoICD. Techniques known to one skilled in the art, such asimmunofluorescence, flow cytometry, and ELISA, can be used to assess theexpression of tumor antigens. In a specific embodiment, the expressionof tumor antigens is assessed as described in Section 6, infra.

In another embodiment, the ability of cryopreserved cancer cellsundergoing ICD after thawing to be phagocytosed by dendritic cells isnot significantly altered as compared to the phagocytosis ofnon-cryopreserved/thawed cancer cells induced to undergo ICD. Techniquesknown to one skilled in the art can be used to assess the phagocytosisof cancer cells undergoing ICD by dendritic cells. In a specificembodiment, the phagocytosis of cancer cells undergoing ICD by dendriticcells is assessed as described in Section 6, infra.

In another embodiment, the ability of cryopreserved cancer cellsundergoing ICD after thawing to induce the maturation of dendritic cellsis not significantly altered relative to the ability ofnon-cryopreserved/thawed cancer cells induced to undergo ICD to inducethe maturation of dendritic cells. In a specific embodiment, thematuration of dendritic cells is assessed by the expression of cellmarkers, such as CD80, CD83 and CD86. Techniques known to one skilled inthe art, such as flow cytometry and FACS, can be used to assess theexpression of such cell markers. In a specific embodiment, thematuration of dendritic cells is assessed as described in Section 6,infra.

In a specific embodiment, dendritic cells pulsed with cryopreservedcancer cells undergoing ICD after thawing are capable of inducingtumor-specific T cells. In another specific embodiment, dendritic cellspulsed with cryopreserved cancer cells undergoing ICD after thawinginduce tumor-specific T cells at levels not significantly different thandendritic cells pulsed with non-cryopreserved/thawed cancer cellsinduced to undergo ICD. Techniques known to one skilled in the art canbe used to assess the induction of tumor-specific T cells. In a specificembodiment, the induction of tumor-specific T cells is assessed asdescribed in Section 6, infra.

5.6 Methods for Generating Dendritic Cells

Techniques known to one skilled in the art may be used toobtain/generate dendritic cells from peripheral blood mononuclear cellsor a bone marrow sample. In a specific embodiment, a whole blood sampleis obtained from a patient and peripheral blood mononuclear cells areisolated. Mononuclear cells may be isolated from peripheral blood of apatient by, for example, Ficoll-Paque Plus gradient centrifugation. Inaddition, mononuclear cells can be fractionated using a fluorescenceactivated cell sorter (FACS) or magnetic activated cell sorting (MACS).Dendritic cells can be enriched by sequential density centrifugation ofapheresis peripheral blood mononuclear cells. The monocytes isolatedfrom the blood of a patient may be cultured in the presence of factors,such as GM-CSF and IL-4 or Flt3L, to differentiate into immature DCsafter a period of, e.g., 4 to 5 days. To generate mature dendriticcells, the cells may be activated with TNF-α, IFN-γ, LPS, CpG, IL-1 orCD40L. In a specific embodiment, mature dendritic cells are activatedusing TLR-3 and/or TLR-4 activators, such as poly (I:C) and/or LPS.Further, CMRF-44 antigen, CD1c, BDCA-4 and other dendritic cell-specificmarkers may also be used to promote DC maturation. In a specificembodiment, dendritic cells are generated as described in Section 6,infra.

Techniques known to one skilled in the art can be used to assess/confirmthe presence of dendritic cells. For example, the presence of dendriticcells can be assessed/confirmed detecting the expression of dendriticcell surface markers using techniques, such as FACS. In a specificembodiment, the presence of dendritic cells is assessed/confirmed usingthe methods in Section 6, infra.

5.7 Methods for Producing Cancer Vaccines

In certain embodiments, dendritic cells are pulsed with the cancer cellsundergoing ICD and the pulsed dendritic cells are the cancer vaccineadministered to a patient. In a preferred embodiment, the cancer cellsundergoing ICD were cryopreserved and thawed before being used to pulsedendritic cells. In a specific embodiment, the pulsed dendritic cellsare autologous to the patient receiving the cancer vaccine.

In some embodiments, dendritic cells are pulsed with the cancer cellsundergoing ICD, the pulsed dendritic cells are co-cultured withlymphocytes (e.g., T lymphocytes) and the lymphocytes are administeredto a patient as the cancer vaccine. In a specific embodiment, the pulseddendritic cells and the lymphocytes are autologous to the patientreceiving the cancer vaccine.

Techniques known to one skilled in the art can be used to pulsedendritic cells with cancer cells undergoing ICD. Further, techniquesknown to one skilled in the art can be used to co-culture lymphocyteswith pulsed dendritic cells.

In a specific embodiment, dendritic cells are pulsed with the cancercells undergoing ICD as described in Section 6, infra.

5.8 Methods for Treating Cancer Using Cancer Vaccines

In a specific aspect, presented herein are methods for treating,protecting against, and/or managing cancer, comprising administering toa subject in need thereof an effective amount of a cancer vaccinedescribed herein or a composition thereof. In a specific embodiment, acancer vaccine described herein or a composition thereof is the onlyactive agent administered to a subject. In certain embodiments, thecancer vaccine described herein that is administered to the subjectcomprises autologous dendritic cells.

In specific embodiments, the administration of a cancer vaccinedescribed herein or a composition thereof to a subject with cancer (insome embodiments, an animal model for cancer) achieves at least one,two, three, four or more of the following effects: (i) the reduction oramelioration of the severity of one or more symptoms of cancer; (ii) thereduction in the duration of one or more symptoms associated withcancer; (iii) the protection against the recurrence of a symptomassociated with cancer; (iv) the reduction in hospitalization of asubject; (v) a reduction in hospitalization length; (vi) the increase inthe survival of a subject; (vii) the enhancement or improvement of thetherapeutic effect of another therapy; (viii) an increase in thesurvival rate of patients; (xiii) a decrease in hospitalization rate;(ix) the protection against the development or onset of one or moresymptoms associated with cancer; (x) the reduction in the number ofsymptoms associated with cancer; (xi) an increase in symptom-freesurvival of cancer patients; (xii) improvement in quality of life asassessed by methods well known in the art; (xiii) the protection againstthe recurrence of a tumor; (xiv) the regression of tumors and/or one ormore symptoms associated therewith; (xvii) the inhibition of theprogression of tumors and/or one or more symptoms associated therewith;(xviii) a reduction in the growth of a tumor; (xix) a decrease in tumorsize (e.g., volume or diameter); (xx) a reduction in the formation of anewly formed tumor; (xxi) eradication, removal, or control of primary,regional and/or metastatic tumors; (xxii) a decrease in the number orsize of metastases; (xxiii) a reduction in mortality; (xxiv) an increasein the tumor-free survival rate of patients; (xxv) an increase inrelapse free survival; (xxvi) an increase in the number of patients inremission; (xxvii) the size of the tumor is maintained and does notincrease or increases by less than the increase of a tumor afteradministration of a standard therapy as measured by conventional methodsavailable to one of skill in the art, such as magnetic resonance imaging(MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, and computedtomography (CT) scan, or a positron emission tomography (PET) scan;(xxviii) an increase in the length of remission in patients; and/or(xxiv) decrease in measurable cancer antigens.

In a specific embodiment, the administration of a cancer vaccinedescribed herein or a composition thereof to a subject with cancer (insome embodiments, an animal model for cancer) inhibits or reduces thegrowth of a tumor by at least 2-fold, preferably at least 2.5-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, or atleast 10-fold relative to the growth of a tumor in a subject with cancer(in some embodiments, in the same animal model for cancer) administereda negative control as measured using assays well known in the art. Inanother embodiment, the administration of a cancer vaccine describedherein or a composition comprising a cancer vaccine described herein toa subject with cancer (in some embodiments, an animal model for cancer)inhibits or reduces the growth of a tumor by at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% relative to the growth of atumor in a subject with cancer (in some embodiments, in the same animalmodel for cancer) administered a negative control as measured usingassays well known in the art.

In a specific embodiment, the administration of a cancer vaccinedescribed herein or a composition comprising a cancer vaccine describedherein to a subject with cancer (in some embodiments, an animal modelfor cancer) reduces the size of a tumor by at least 2-fold, preferablyat least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 7-fold, or at least 10-fold relative to the growth of a tumor in asubject with cancer (in some embodiments, the same animal model forcancer) administered a negative control as measured using assays wellknown in the art. In another embodiment, the administration of a cancervaccine described herein or a composition comprising a cancer vaccinedescribed herein to a subject with (in some embodiments, an animal modelfor cancer) reduces the size of a tumor by at least 10%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95%, or 10% to 25%,25% to 50%, 25% to 75%, 50% to 75%, 75% to 100% relative to the growthof a tumor in a subject with cancer (in some embodiments, the sameanimal model for cancer) administered a negative control as measuredusing assays well known in the art.

In some embodiments, a cancer vaccine described herein is administeredto a subject in combination with one or more other therapies, e.g.,anti-cancer agents, cytokines, cellular vaccines or anti-hormonalagents, to treat and/or manage cancer. In one embodiment, thecombination of a cancer vaccine described herein and one or more othertherapies provides an additive therapeutic effect relative to thetherapeutic effects of the cancer vaccine described herein alone or theone or more other therapies alone. In another embodiment, thecombination of a cancer vaccine described herein and one or more othertherapies provides more than an additive therapeutic effect relative tothe therapeutic effects of the cancer vaccine described herein alone orthe one or more other therapies alone. In a specific embodiment, thecombination of a cancer vaccine described herein and one or more othertherapies provides a synergistic therapeutic effect relative to thetherapeutic effects of the cancer vaccine described herein alone or theone or more other therapies alone.

In a specific embodiment, a cancer vaccine described herein isadministered in combination with radiation therapy comprising, e.g., theuse of x-rays, gamma rays and other sources of radiation to destroy thecancer cells. In specific embodiments, the radiation treatment isadministered as external beam radiation or teletherapy wherein theradiation is directed from a remote source. In other embodiments, theradiation treatment is administered as internal therapy or brachytherapywherein a radioactive source is placed inside the body close to cancercells or a tumor mass. In one aspect, the cancer vaccine describedherein can activate or enhance the immune function of cancer patientwith a compromised immune system due to anti-cancer therapy. In anotherembodiment, a cancer vaccine described herein is administered incombination with chemotherapy. In an embodiment, a cancer vaccinedescribed herein can be used before, during or after radiation therapyor chemotherapy. In another embodiment, a cancer vaccine describedherein can be used before, during or after surgery.

5.8.1 Types of Cancers

Cancers and related disorders that can be treated, protected against, ormanaged in accordance with the methods described herein include, but arenot limited to, the following: leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic Leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, and chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, and non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, and primary brain lymphoma; breast cancer including, butnot limited to, adenocarcinoma, lobular (small cell) carcinoma,intraductal carcinoma, medullary breast cancer, mucinous breast cancer,tubular breast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytoma and adrenocortical carcinoma; thyroid cancer suchas but not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma; gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, semi noma, anaplastic,spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma,choriocarcinoma (yolk-sac tumor); prostate cancers including but notlimited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penalcancers; oral cancers including but not limited to, squamous cellcarcinoma; basal cancers; salivary gland cancers including but notlimited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcysticcarcinoma; pharynx cancers including but not limited to, squamous cellcancer, and verrucous; skin cancers including but not limited to, basalcell carcinoma, squamous cell carcinoma and melanoma, and superficialspreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers including but not limited to, renalcell cancer, renal cancer, adenocarcinoma, hypernephroma, fibrosarcoma,and transitional cell cancer (renal pelvis and/or uterer); Wilms' tumor;bladder cancers including but not limited to, transitional cellcarcinoma, squamous cell cancer, adenocarcinoma, and carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

In one embodiment, the cancer is benign, e.g., polyps and benignlesions. In other embodiments, the cancer is metastatic. The cancervaccine can be used in the treatment of pre-malignant as well asmalignant conditions. Pre-malignant conditions include hyperplasia,metaplasia, and dysplasia. Treatment of malignant conditions includesthe treatment of primary as well as metastatic tumors. In oneembodiment, the cancer is a solid tumor. In a specific embodiment thecancer is melanoma, colon cancer, prostate cancer, ovarian cancer,pancreatic cancer, lung cancer, rhabdomyosarcoma, neuroblastoma, Ewingsarcoma, gastric cancer, or hepatoma.

5.8.2 Patient Populations

In some embodiments, a cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapiesare administered to a subject suffering from or diagnosed with cancer.In other embodiments, a cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapiesare administered to a subject predisposed or susceptible to developingcancer. In some embodiments, a cancer vaccine described herein,compositions comprising a cancer vaccine described herein, orcombination therapies are administered to a subject that lives in aregion where there is a high occurrence rate of cancer. In a specificembodiment, the cancer is characterized by a pre-malignant tumor or amalignant tumor.

In some embodiments, a cancer vaccine described herein, compositioncomprising a cancer vaccine described herein, or a combination therapyis administered to a mammal. In certain embodiments, a cancer vaccinedescribed herein, composition comprising a cancer vaccine describedherein, or a combination therapy is administered to a mammal which is 0to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 yearsold, 10 to 15 years old, 15 to 20 years old, 20 to 25 years old, 25 to30 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 yearsold, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 yearsold, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to100 years old. In certain embodiments, a cancer vaccine describedherein, composition comprising a cancer vaccine described herein, or acombination therapy is administered to a pet, e.g., a dog or cat. Incertain embodiments, a cancer vaccine described herein, compositioncomprising a cancer vaccine described herein, or a combination therapyis administered to a farm animal or livestock, e.g., pig, cows, horses,chickens, etc.

In certain embodiments, a cancer vaccine described herein, compositioncomprising a cancer vaccine described herein, or a combination therapyis administered to a human at risk developing cancer. In certainembodiments, a cancer vaccine described herein, composition comprising acancer vaccine described herein, or a combination therapy isadministered to a human with cancer. In certain embodiments, a cancervaccine described herein, composition comprising a cancer vaccinedescribed herein, or a combination therapy is administered to a humandiagnosed with cancer. In certain embodiments, the patient is a human 0to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 yearsold, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13 to 19years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 years old,30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old,65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 yearsold.

In certain embodiments, a cancer vaccine described herein, compositioncomprising a cancer vaccine described herein, or a combination therapyis administered to a subject that has or is at risk of getting AIDS, aviral infection, or a bacterial infection. In certain embodiments, asubject that is, will or has undergone surgery, chemotherapy and/orradiation therapy.

In some embodiments, a patient is administered a cancer vaccinedescribed herein, composition comprising a cancer vaccine describedherein, or a combination therapy is before any adverse effects orintolerance to therapies other than a cancer vaccine described hereindevelops. In some embodiments, a cancer vaccine described herein,compositions comprising a cancer vaccine described herein, orcombination therapies are administered to refractory patients. In acertain embodiment, refractory patient is a patient refractory to astandard anti-cancer therapy. In certain embodiments, a patient withcancer is refractory to a therapy when the cancer has not significantlybeen eradicated and/or the symptoms have not been significantlyalleviated. The determination of whether a patient is refractory can bemade either in vivo or in vitro by any method known in the art forassaying the effectiveness of a treatment, using art-accepted meaningsof “refractory” in such a context. In various embodiments, a patientwith cancer is refractory when a cancerous tumor has not decreased orhas increased.

In some embodiments, a cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapiesare administered to a patient to protect against the onset orreoccurrence of cancer in a patient at risk of developing such cancer.In some embodiments, a cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapiesare administered to a patient who is susceptible to adverse reactions toconventional therapies.

In some embodiments, a cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapiesare administered to a patient who has proven refractory to therapiesother than a cancer vaccine described herein, but are no longer on thesetherapies. In certain embodiments, the patients being managed or treatedin accordance with the methods described herein are patients alreadybeing treated with antibiotics, anti-cancer agents, or other biologicaltherapy/immunotherapy. Among these patients are refractory patients,patients who are too young for conventional therapies, and patients withreoccurring viral infections despite management or treatment withexisting therapies.

In some embodiments, the subject being administered a cancer vaccinedescribed herein, compositions comprising a cancer vaccine describedherein, or combination therapies has not received a therapy prior to theadministration of the cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapies.In other embodiments, a cancer vaccine described herein, compositionscomprising a cancer vaccine described herein, or combination therapiesare administered to a subject who has received a therapy prior toadministration of one or more a cancer vaccine described herein,compositions comprising a cancer vaccine described herein, orcombination therapies. In some embodiments, the subject administered acancer vaccine described herein or a composition comprising a cancervaccine described herein was refractory to a prior therapy orexperienced adverse side effects to the prior therapy or the priortherapy was discontinued due to unacceptable levels of toxicity to thesubject.

5.9 Administration and Dosage

5.9.1 Mode of Administration

A cancer vaccine described herein or composition thereof can beadministered via any route known in the art. A cancer vaccine describedherein or compositions thereof can be administered by, for example,infusion or bolus injection, and may be administered together withanother biologically active agent. Administration can be systemic orlocal. Various delivery systems are known and can be used to deliver acancer vaccine described herein or compositions thereof.

Methods of administration include but, are not limited to, parenteral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intracerebral, or intratumorally. In a specific embodiment, the cancervaccine is intravenously, intradermally or subcutanouesly administeredto the patient. In another specific embodiment, the cancer vaccine isadministered to the patient by direct intranodal delivery. In anotherspecific embodiment, the cancer vaccine is administered to the tumoritself. The mode of administration is left to the discretion of thepractitioner.

In specific embodiments, it may be desirable to administer a cancervaccine or composition thereof locally. This may be achieved, forexample, and not by way of limitation, by local infusion or by means ofan implant, said implant being of a porous or gelatinous material,including membranes, such as sialastic membranes, or fibers.

5.9.2 Dosage

The amount of a cancer vaccine described herein, or the amount of acomposition comprising a cancer vaccine described herein, that will beeffective in the treatment of, protection against, and/or management ofcancer can be determined by standard clinical techniques. In vitro or invivo assays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed will also depend, e.g., on theroute of administration, the type of symptoms, and the seriousness ofthe symptoms, and should be decided according to the judgment of thepractitioner and each patient's or subject's circumstances.

Doses of pulsed-dendritic cells described herein for administration byany route of administration can be at least 100, 200, 300, 400, 500,700, 1,000, 5,000, 10,000, 25,000, 50,000, or 100,000 cells. In specificembodiments, the number of pulsed-dendritic cells is at least 100, 200,300, 400, 500 cells. In other embodiments, the number ofpulsed-dendritic cells is at least 300, 400, 500, 700, 1,000 cells. Inyet other specific embodiments, the number of pulsed-dendritic cells isat least 700, 1,000, 5,000, 10,000 cells. In some embodiments, thenumber of pulsed-dendritic cells is at least 5,000, 10,000, 25,000,50,000, or 100,000 cells. In yet another embodiment, the number of cellsis at least 50,000, or 100,000 cells. In other embodiments, the numberof pulsed-dendritic cells is at least 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸,5×10⁸ or more cells. In specific embodiments, the number ofpulsed-dendritic cells is between 1×10² to 1×10⁴, 5×10⁴ to 5×10⁶, 1×10⁵to 1×10⁷, 1×10⁵ to 5×10⁸, 1×10⁶ to 1×10⁸, or 1×10⁶ to 1×10⁷, or 1×10⁴ to1×10⁵ cells.

In certain embodiments, a subject is administered a cancer vaccinedescribed herein or composition thereof in an amount effective toinhibit or reduce symptoms associated with cancer by at least 20% to25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35%to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%,at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least70% to 75%, at least 75% to 80%, or up to at least 85% relative to anegative control as determined using an assay described herein or othersknown to one of skill in the art. In certain embodiments to treat, asubject is administered a cancer vaccine described herein or acomposition thereof in an amount effective to inhibit or reduce symptomsassociated with cancer by at least 1.5-fold, 2-fold, 2.5-fold, 3-fold,4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or 2- to 5-fold, 2-to 10-fold, 5- to 10-fold, or 5- to 20-fold relative to a negativecontrol as determined using an assay described herein or other known toone of skill in the art.

In certain embodiments, to treat, protect against, and/or manage cancer,a subject is administered a cancer vaccine described herein orcomposition thereof in an amount effective to inhibit or reduce tumorgrowth or cancer cell proliferation by at least 20% to 25%, preferablyat least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, atleast 75% to 80%, or up to at least 85% relative to a negative controlas determined using an assay described herein or others known to one ofskill in the art. In some embodiments, a subject is administered acancer vaccine described herein or composition thereof in an amounteffective to inhibit or reduce tumor growth or cancer cell proliferationby at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 8-fold,10-fold, 15-fold, 20-fold, or 2 to 5-fold, 2 to 10-fold, 5 to 10-fold,or 5 to 20-fold relative to a negative control as determined using anassay described herein or others known to one of skill in the art.

In certain embodiments to, a subject is administered a cancer vaccinedescribed herein or composition thereof in an amount effective to induceor enhance an immune response by at least 20% to 25%, preferably atleast 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40%to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%,at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least75% to 80%, or up to at least 85% relative to a negative control asdetermined using an assay described herein or others known to one ofskill in the art. In some embodiments, a subject is administered acancer vaccine described herein or composition thereof in an amounteffective to induce or enhance an immune response by at least 1.5-fold,2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold,20-fold, or 2 to 5-fold, 2 to 10-fold, 5 to 10-fold, or 5 to 20-foldrelative to a negative control as determined using an assay describedherein or others known to one of skill in the art.

In certain embodiments to, a subject is administered a cancer vaccinedescribed herein or composition thereof in an amount effective toincrease or enhance the number of lymphocytes (in some embodiments, in aspecific target body compartment) by at least 20% to 25%, preferably atleast 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40%to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%,at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least75% to 80%, or up to at least 85% relative to a negative control asdetermined using an assay described herein or others known to one ofskill in the art. In some embodiments, a subject is administered acancer vaccine described herein or composition thereof in an amounteffective to increase or enhance the number of lymphocytes (in someembodiments, in a specific target body compartment) by at least1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least15-fold, or at least 20-fold; or by approximately 2 to 5-fold, 2 to10-fold, 5 to 10-fold, or 5 to 20-fold relative to a negative control asdetermined using an assay described herein or others known to one ofskill in the art. In some embodiments, the specific target bodycompartment where the number of lymphocytes is increased or enhanced bya cancer vaccine described herein is the lung, stomach, heart, kidney,liver, small intestines, large intestines, breast, prostate, or bladder.In particular embodiments, the specific target body compartment wherethe number of lymphocytes is increased or enhanced is the bodycompartment affected by cancer. In some embodiments, the specific targetbody compartment where the number of lymphocytes is increased orenhanced is the lymph node, spleen, or peripheral blood.

In certain embodiments, a dose of a cancer vaccine described herein orcomposition thereof is administered to a subject every day, every otherday, every couple of days, every third day, once a week, twice a week,three times a week, or once every two weeks or once a month, or less. Inother embodiments, two, three or four doses of a cancer vaccinedescribed herein or composition thereof is administered to a subjectevery day, every couple of days, every third day, once a week or onceevery two weeks. In some embodiments, a dose(s) of a cancer vaccinedescribed herein or composition thereof is administered for 2 days, 3days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, adose of a cancer vaccine described herein or composition thereof isadministered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4months, 5 months, 6 months or more.

In a preferred embodiment, a patient is administered multiple doses of acancer vaccine, wherein each dose of the cancer vaccine comprisesdendritic cells pulsed with cancer cells undergoing ICD which had beencryopreserved and thawed. In a specific embodiment, a batch of dendriticcells (which had been pulsed with cancer cells which had been induced toundergo ICD and cryopreserved) is frozen and stored in separatecontainers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more containers, such as vials) and one container suchas a vial, or optionally more than 1 container, is thawed for deliveryof the cancer vaccine to a patient. This allows for standardization ofthe manufacturing process for the dendritic cell vaccine so that two ormore patients may be treated with vaccines produced from the same batchof cryopreserved cancer cells undergoing ICD.

The dosages of prophylactic or therapeutic agents which have been or arecurrently used for the treatment of, protection against, and/ormanagement of cancer can be determined using references available to aclinician such as, e.g., the Physicians' Desk Reference (67th ed. 2013).In a specific embodiment, dosages lower than those which have been orare currently being used to treat, protect against, and/or manage cancerare utilized in combination with a cancer vaccine described herein orcompositions thereof.

The above-described administration schedules are provided forillustrative purposes only and should not be considered limiting.

5.10 Biological Assays

The antigen presenting capability of pulsed dendritic cells can beassessed using techniques known to one skilled in the art. Variousassays known in the art can be used to assess whether a cancer vaccinedescribed herein activates or enhances an immune function. In oneaspect, a cancer vaccine described herein increases an immune responsethat can be, e.g., an antibody response (humoral response) or a cellularimmune response, e.g., cytokine secretion (e.g., interferon), chemokinesecretion, helper activity or cellular cytotoxicity. In a specificembodiment, the ability of pulsed dendritic cells to induce IFN-gammaproduction of lymphocytes is assessed.

Proliferation of certain immune cells (e.g., lymphocytes) may beassessed by ³H-thymidine incorporation. The cytotoxicity of T cells canbe tested in a ⁵¹Cr-release assay as described in the art.

An ELISPOT assay can be used to measure cytokine release by lymphocytesco-cultured by pulsed dendritic cells described herein. Cytokinesecretion can be detected by antibodies which are specific for aparticular cytokine, e.g., interleukin-2, tumor necrosis factor-α orinterferon-γ, or chemokine. In a specific embodiment, a cytokinesecretion by a cancer vaccine can be assessed using the techniquesdescribed in Section 6, infra.

In specific embodiments, a cancer vaccine described herein induces orenhances lymphocyte cell proliferation in a subject that by at least 0.2to 5 times, 5 to 20 times, 10 to 30 times, 20 to 50 times, 50 to 200times, 100 to 500, 200 to 1000 times, or 500 to 2,000 times higherrelative to lymphocyte cell proliferation in a negative control. Inspecific embodiments, a cancer vaccine described herein induces orenhances T cell proliferation in a subject that by at least 0.2 to 5times, 5 to 20 times, 10 to 30 times, 20 to 50 times, 50 to 200 times,100 to 500, 200 to 1000 times, or 500 to 2,000 times higher relative toT cell proliferation in a negative control as determined by methods wellknown in the art, e.g., flow cytometry, CSFE staining, ³H-thymidineincorporation.

5.11 Methods for Producing Vaccines for Immunotherapy

In certain embodiments, dendritic cells are pulsed with the animal cellsthat express an antigen(s) of interest and are undergoing ICD, and thepulsed dendritic cells are the vaccine administered to a patient. In apreferred embodiment, the cells undergoing ICD were cryopreserved andthawed before being used to pulse dendritic cells. In a specificembodiment, the pulsed dendritic cells are autologous to the patientreceiving the vaccine.

In some embodiments, dendritic cells are pulsed with the animal cellsthat express an antigen(s) of interest and are undergoing ICD, thepulsed dendritic cells are co-cultured with lymphocytes (e.g., Tlymphocytes) and the lymphocytes are administered to a patient as thevaccine. In a specific embodiment, the pulsed dendritic cells and thelymphocytes are autologous to the patient receiving the cancer vaccine.

Techniques known to one skilled in the art can be used to pulsedendritic cells with cancer cells undergoing ICD. Further, techniquesknown to one skilled in the art can be used to co-culture lymphocyteswith pulsed dendritic cells.

5.12 Kits

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers comprising cryopreserved cells undergoing ICD. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical pack or kit may include instructions foruse of the cryopreserved cells described herein. The kits encompassedherein can be used in the above methods.

In a specific embodiment, a pharmaceutical pack or kit comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morecontainers (e.g., vials) containing cryopreserved cancer cellsundergoing ICD. In a particular embodiment, each container containscryopreserved cancer cells all from the same batch of cancer cellsinduced to undergo ICD. In certain embodiments, each container (e.g., avial) of cryopreserved cancer cells contains 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000cancer cells induced to undergo ICD. In some embodiments, each container(e.g., a vial) of cryopreserved cancer cells contains 1×10², 5×10²,1×10³, 5×10³, 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷ or1×10⁸ cancer cells induced to undergo ICD. In certain embodiments, eachcontainer (e.g., a vial) of cryopreserved cancer cells contains 1×10¹ to1×10², 1×10² to 5×10², 1×10² to 1×10³, 1×10³ to 5×10³, 1×10² to 1×10⁴,1×10³ to 5×10⁴, 1×10³ to 1×10⁴, 1×10² to 1×10⁵, 1×10² to 5×10⁵, 1×10² to1×10⁶, 1×10³ to 5×10⁶, 1×10⁴ to 1×10⁷, 1×10⁵ to 1×10⁷, 1×10⁵ to 1×10⁷,1×10⁶ to 1×10⁸, 1×10⁷ to 1×10⁸ cancer cells induced to undergo ICD.

In another embodiment, approximately 55%, approximately 60%,approximately 65%, approximately 70%, approximately 75%, approximately80%, approximately 85%, approximately 90%, approximately 95% orapproximately 98% of cryopreserved cancer cells undergoing ICD in acontainer (e.g., a vial) maintain one, two, three or more, or all of thecharacteristics and/or functions of non-cryopreserved/thawed cancercells induce to undergo ICD after thawing as assessed by techniquesdescribed herein (e.g., in Section 5.2, 5.5, and 6) and/or known to oneskilled in the art. In another specific embodiment, approximately 55%,approximately 60%, approximately 65%, approximately 70%, approximately75%, approximately 80%, approximately 85%, approximately 90%,approximately 95% or approximately 98% of the cryopreserved cancer cellsin a container (e.g., a vial) express ICD markers after thawing. In someembodiments, approximately 55% to approximately 65%, approximately 65%to approximately 85%, or approximately 75%, to approximately 85%,approximately 80% to approximately 90% or approximately 90% toapproximately 95% of cryopreserved cancer cells undergoing ICD in acontainer (e.g., a vial) maintain one, two, three or more, or all of thecharacteristics and/or functions of non-cryopreserved/thawed cancercells induce to undergo ICD after thawing as assessed by techniquesdescribed herein (e.g., in Section 5.2, 5.5, and 6) and/or known to oneskilled in the art.

In a specific embodiment, at least 50% of the cryopreserved cancer cellsin a container (e.g., a vial) express markers of ICD after thawing. Inspecific embodiments, approximately 55%, approximately 60%,approximately 65%, approximately 70%, approximately 75%, approximately80%, approximately 85%, approximately 90%, approximately 95% orapproximately 98% of the cryopreserved cancer cells in a container(e.g., a vial) express ICD markers after thawing. In some embodiments,approximately 55% to approximately 65%, approximately 65% toapproximately 85%, or approximately 75%, to approximately 85%,approximately 80% to approximately 90% or approximately 90% toapproximately 95% of the cryopreserved cancer cells in a container(e.g., a vial). In a specific embodiment, the expression of ICD markers,such as HSP70, HSP90 and calreticulin, by cryopreserved cancer cellsundergoing ICD after thawing is not significantly altered relative tonon-cryopreserved/thawed cancer cells induced to undergo ICD.

6. EXAMPLES

This example demonstrates that HHP treated and cryopreserved cancercells are a reliable and potent source of tumor antigens forimmunotherapy protocols. The HHP-frozen cancer cell death fulfills allcurrently described criteria of immunogenic cell death, including thepreferentially activation of apoptotic pathway. The HHP-frozen cancercells carry sufficient amount of tumor associated antigens, such as PSA,PSMA, and Her2/Neu. Further, the HHP-frozen cancer cells are able toactivate dendritic cells and induce an extended level of antigenspecific T cell response.

6.1 Materials & Methods

Cell Line:

Prostate cancer cells (LNCap; HLA-A2 positive; ATCC) and ovarian cancercells (SKOV3; HLA-A2 positive; ATCC) were cultured in RPMI 1640 medium(Gibco). Medium was supplemented with 10% heat-inactivated fetal bovineserum (PAA), 100 U/ml penicillin and 2 mmol/L L-glutamine.

Antibodies and Reagents:

The following monoclonal antibodies (mAbs) against the indicatedmolecules were used: CD80-FITC, CD83-PE, CD86-PE-Cy5, CD14-PE-Dy590,CD8-PE-Dy590 (Exbio), CD11c-PE, HLA-DR-Alexa700, IFNγ-FITC, CD4-PC7 (BDBioscineces), anti-HSP70 (R&D), anti-HSP90, anti-calreticulin (Enzo),anti-PSA, GAPDH (Genetex), anti-PSMA (Abcam) and anti-Her2/Neu(Genetex).

Apoptosis Induction and Detection:

Tumor cell death was induced by HHP treatment (250 MPa, 10 mins.). Celldeath was assessed by annexin V fluorescein isothiocyanate stainingBriefly, 2×10⁵ cells per sample were collected, washed in PBS, pelleted,and resuspended in an incubation buffer containing annexin V Dy647(Exbio). The samples were kept in the dark and incubated for 15 minbefore the addition of DAPI and subsequent analysis on FACS Fortessa (BDBioscience) using FlowJo software.

Flow Cytometric Analysis of HSP70, HSP90 and CRT:

A total of 1×10⁵ cells were plated in 12-well plates and treated by HHPfor 6, 12 or 24 h. The cells were collected and washed twice with PBS.The cells were incubated for 30 min with primary antibody, followed bywashing and incubation with the Alexa 648-conjugated monoclonalsecondary antibody in a blocking solution. Each sample was then analyzedby FACScan Fortessa (BD Bioscience) to identify expression of HSP70,HSP90 and CRT.

Immunofluorescence of HSP70, HSP90 and CRT by Confocal Microscopy:

The cells were collected and washed twice with PBS. The cells were thenincubated for 30 min with primary antibody diluted in cold blockingbuffer (2% fetal bovine serum in PBS), followed by washing andincubation with the Alexa Fluor 488 goat anti-mouse secondary antibody.Cells were washed twice with PBS and fixed in 4% paraformaldehyde in PBSfor 20 min and mounted on slides. The emission spectra of green HSP70,HSP90 or CRT were detected in the 500-550 nm range. Cells were examinedunder a DMI 6000 inverted Leica TCS AOBS SP5 tandem scanning confocalmicroscope with an AR (488 nm) laser and an x63 oil immersion objective.

Generation of Dendritic Cells:

Immature monocyte-derived DCs (moDCs) were generated as previouslydescribed. Briefly, peripheral blood mononuclear cells (PBMCs) wereisolated from buffy coats of healthy HLA-A2⁺ or HLA-A2⁻ donors byFicoll-Paque PLUS gradient centrifugation (GE Healthcare, Uppsala,Sweden) and monocytes were isolated by plastic adherence after 2 h ofcell adhesion (75×10⁶ PBMCs) in Nunclon 75-cm2 culture flasks (Nunc).Adherent monocytes were subsequently cultured for 6 days in serum freeCellGro DC media (CellGenix) in the presence of GM-CSF (Gentaur) at aconcentration of 500 U/ml and 20 ng/ml of IL-4 (Gentaur). After 3 daysof culture, fresh CellGro and cytokines were added to the cultureflasks. After 6 days, immature DCs were seeded in Nunclon 48-well plates(5×10⁵ DCs in 500 μl of CellGro supplemented with cytokines per well)and pulsed with apoptotic LNCap cells for 4 h, furthermore DC werematurated by Poly (I:C) (InvivoGen) at 25 μg/ml or LPS (Sigma-Aldrich)at 1 μg/ml. Immature and mature DCs were used for further studies. Forcocultures, a fraction of the PBMCs was cryopreserved, thawed and usedfor generation of DCs for restimulation. Non-adherent monocyte-depletedPBMCs were frozen and used as lymphocytes for cocultures with DCs.

Flow Cytometry:

Immature and mature DCs were phenotyped using the following monoclonalantibodies: CD80-FITC, CD86-PE, CD83-PE-Cy5 (Beckman Coulter),CD14-PE-Dy590, CD11c-APC (Exbio) and HLA-DR-PE-Cy7 (BD Biosciences). Thecells were stained for 20 min at 4° C., washed twice in PBS and analyzedusing LSRFortessa (BD Biosciences) with FlowJo software (Tree Star). DCswere gated according to their FSC and SSC properties and as CD11cpositive cells. Only viable DCs (DAPI negative cells) were included inthe analysis. DAPI was purchased from Invitrogen.

FACS Analysis of DC Phenotype after Interaction with Killed Tumor Cells:

The phenotype of DCs cultured with tumor cells was monitored by flowcytometry. Tumor cells were killed by HHP and were cocultured for 24 hwith immature DCs. For some experiments, the DCs and tumor cells weredye-labeled before coculture to monitor phagocytosis. Monoclonalantibodies (mAbs) against the following molecules were used: CD80-A700(Exbio), CD83-PerCP-Cy5,5 (BioLegend), CD86-A647 (BioLegend), CD14-PE(Exbio), CD11c-APC (Exbio), HLA-DR PC7 (BD Biosciences).

The DCs were stained for 30 minutes at 4° C., washed twice inphosphate-buffered saline (PBS) and analyzed using FACS Aria (BDBiosciences) using FlowJo software. The DCs were gated according to theFSC and SSC properties. The appropriate isotype controls were included,and 50000 viable DCs were acquired for each experiment.

Uptake of HHP-Treated Cancer Cells by DCs:

For flow cytometry analysis of phagocytosis, tumor cells were harvestedand labeled with Vybrant DiD cell labeling solution (Invitrogen). Toprepare HHP treated cells, stained LNCap cells were seeded in CellGromedia in Nunclon 25-cm² culture flasks (Nunc) at a concentration of4×10⁵ cells/ml and subjected to a 250 MPa of HHP for 10 minutes toinduce apoptosis. Cells were then incubated for 24 h at 37° C. with 5%CO2 before use. To determine the uptake of HHP-treated tumor cells byDCs, immature DCs were stained with Vybrant DiO cell labeling solution(Invitrogen) and cocultured with LNCap cells at a cell ratio of 5:1 inNunclon U-bottom 96-well plates (Nunc) for 24 h at 37° C. with 5% CO₂.Parallel control cultures were set up for 24 h on ice to evaluate thepassive transfer of dye or labeled tumor fragments to DCs. Thephagocytic ability of DCs was evaluated by flow cytometry.

Expansion of Antigen-Specific T-Lymphocytes and Intracellular IFN-γStaining:

Immature DCs were fed tumor cells at a DC/tumor cell ratio of 5:1 for 24hours. In some experiments pulsed DCs were stimulated with Poly (I:C)(25 μg/ml). Non adherent peripheral blood lymphocytes (PBL) (2×10⁵ inRPMI-1640+10% AB human serum (Invitrogen)) and the mature pulsed DCs(4×10⁴ in CellGro) were cocultured at a ratio of 5:1 in U-bottom 96-wellplates for 7 days. A total of 20 U/ml of IL-2 (PeproTech) was added ondays 3 and 5. On day 7, the lymphocytes were restimulated with freshtumor cells pulsed DCs, and the frequency of antigen-specific T cellswas determined using intracellular staining for IFN-γ. Brefeldin A(BioLegend) was added to block the extracellular release of IFN-γ onehour and a half after restimulation. After 3 h of incubation withBrefeldin A, the cells were washed in PBS, stained withanti-CD3-PerCP-Cy5.5 (eBioscience) and CD8-PE-Dy590 antibody (Exbio),fixed using Fixation Buffer (eBioscience), permeabilized withPermeabilization Buffer (eBioscience) and stained using anti-IFN-γ-FITCantibody (BD Biosciences). The cells were acquired using the LSRFortessa(BD Biosciences) and analyzed with FlowJo software (Tree Star).

Preparation of Cell Extracts and Western Immunoblot Analysis:

Cell extracts were prepared at the indicated time points following HHPtreatment. After treatment cells were washed with ice-cold PBS and lysedon ice in RIPA buffer (10 mM TRIS pH7.5, 150 mM NaCl, 5 mM EDTA, 1%Triton X 100)+protease inhibitor cocktail (Roche Diagnostics) and 1 mMPMSF (phenylmethylsulfonyl fluoride). Proteins were separated by 12%SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). Themembranes were blocked in 5% nonfat dry milk in TBST buffer (50 mM Tris,150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature and incubatedwith primary antibody overnight at 4° C. The membranes were then washedin TBST and incubated for 1 h at room temperature with horseradishperoxidase-conjugated secondary antibodies. Detection was carried outwith the enhanced chemiluminescence (ECL) detection system. Equalprotein loading was ensured by BCA assay, verified by analysis ofPonceau-S staining of the membrane and GADPH reprobing.

Statistical Analysis:

The data were analyzed by One-Way ANOVA with Dunnett's multiplecomparison post hoc test and Student's unpaired two-tailed t test usingGraphPad Prism 5 (San Diego, Calif., USA). The results were consideredstatistically significant when p<0.05.

6.2 Results

Cryopreservation of HHP Treated Tumor Cells Preserves the ApoptoticCharacter of Cell Death and the Expression of Immunogenic Cell DeathMarkers:

To initially determine the ability of HHP-treated and frozen tumor cellsto preserve features of immunogenic cell death, the prostate cancer cellline LNCap and ovarian cancer cell line SKOV3 were used. To verifywhether HHP treated frozen cancer cells die by apoptosis or necrosis,tumor cells were treated by 250 MPa for 10 min and frozen for minimalcourse of 1 week and thawed. Cell death was analyzed according to DAPIand annexin V staining (FIG. 1A, B). Treatment of both LNCap and SKOV3cells with HHP led to the apoptosis (% annexin V+ cells) of more than50% immediately after the treatment with the majority of cellsdemonstrating a staining pattern typical of apoptosis (annexin V+)rather than necrosis (annexinV−/DAPI+) (FIG. 1B). Furthermore, theapoptosis of frozen-HHP cells was evaluated. Cells frozen in time OH and4H after the HHP-treatment and determined in OH and 4H timepoints aredying preferentially in apoptotic way with more than 90% of AnnexinV+/DAPI+ cells (FIG. 1A, B).

Expanded tumor cells were collected from culture flasks by usingtrypsin. Detached cells were centrifuged and washed with PBS+EDTAmultiple times. The viability of the cells was assessed by microscopyusing a 0.4% trypan blue solution. Cells were resuspended in growthmedia and immunogenic cell death was induced by HHP. Following HHP thestate of the cells was assessed by microscopy and incubated in growthmedia for 2 hours in a CO₂ incubator at 37° C. After incubation, thecells were collected, washed, resuspended in freezing medium CryoStorCS10 and aliquoted in freezing vials (20×10⁶ cells for vial, 1 ml pervial). Freezing was done by placing the vials at −80° C. overnight in aisopropanol container to ensure slow freezing. The next day the vialswere transferred into a liquid nitrogen tank. Cells were thawed prior toincubation with DCs. The cells were thawed in the palm of the hand or inan incubator at 37° C. in order to assure a quick thaw and resuspendedin warm growth media.

The kinetics of the expression of immunogenic cell death markers ontumor cells undergoing apoptosis induced by HHP and HHP-frozen in timeOH and 4H was also examined. Treatment of tumor cells with HHP 250 MPafor 10 min led to the significant expression of HSP70, HSP90 and CRTcompared to untreated cells. Additionally, tumor cells treated by HHPand frozen in time OH or 4H were expressing the immunogenic molecules inincreased level compared to untreated cells but also significantlyhigher to HHP treated non frozen cells (FIG. 1C). To verify the presenceof immunogenic markers on cell surface, the confocal microscopy for CRT(FIG. 1E) and HSP70 and HSP90 (data not shown) was performed.

HHP-Frozen Tumor Cells are Preserving the Immunogenicity in Long TimeCourse:

After the initial examination of the presence of immunogenic cell deathmarkers after HHP treatment and freezing process, the stability ofexpression of these markers in long time course (2 weeks, 1, 3 and 6months) was evaluated. Expression of HSP70, HSP90 and CRT by othercancer cell lines are detected to a similar extent for 6 months (FIG.1D).

HHP Treatment and Cryopreservation of Tumor Cells Induce Positively anAccumulation of Tumor Antigens Compared to Other Cytoskeletal Proteins:

Next, the presence of the two most expressed tumor antigens on the cellsurface of prostate LNCap cell line PSA (prostate-specific antigen) andPSMA (prostate-specific membrane antigen) and the expression of Her2/Neutumor antigen on the cell surface of SKOV3 cell line were analyzed byflow cytometry after the HHP treatment and in HHP-frozen tumor cells.Treatment of tumor cells by HHP does not influence the expression levelof any mentioned tumor antigen (PSA, PSMA and Her2/Neu) (FIG. 2A).HHP-frozen tumor cells are expressing the same level of all threeantigens tested in all tested times and conditions. To determine thewhole amount of protein PSA and PSMA in LNCap cell line and Her2/Neu inSKOV3 cell line after the treatment of HHP and cryopreservation, WesternBlot analysis was used. Compared to untreated cells, cells treated withHHP have a higher detected amount of PSA and PSMA protein (FIG. 2B, C).Furthermore, after the cryopreservation, the protein level of PSA, PSMAand Her2/Neu was significantly higher than those in untreated cells andeven significantly higher than in HHP treated but non frozen cells.

HHP-Frozen Tumor Cells are Phagocytosed by DCs at the Same Level asNon-Frozen Cells:

In view of the established role of CRT as a phagocytosis-promotingsignal, the rate of phagocytosis of HHP-treated and HHP-frozen prostate(LNCap) and ovarian (SKOV3) tumor cells by DCs by flow cytometry wasinvestigated. As previously described, HHP-treated tumor cells arephagocytosed at a faster rate and to a greater extent than tumor cellskilled by modifications like UV-B irradiation, photodynamic therapy byhypericin or anthracyclines. After 24 h, the rate of phagocytosis ofHHP-frozen tumor cells was non-significantly higher in comparison tonon-cryopreserved HHP treated cells (FIG. 3A, B).

Phagocytosis of HHP-Frozen Tumor Cells Induces the Expression ofMaturation-Associated Molecules on DCs:

The ability of DCs to activate the immune response depends on theirmaturation status and the expression of costimulatory molecules. Thephenotype of DCs that had phagocytosed prostate (LNCap) and ovarian(SKOV3) tumor cells killed by HHP and HHP-frozen was analyzed. The datademonstrates that the interaction of DCs with HHP-frozen tumor cellsinduce the same significant upregulation of CD80, CD83, and CD86 withsubsequent maturation with polyI:C. Thus, cryopreservation of tumorcells does not negatively influence the maturation process of dendriticcells. Furthermore, cryopreservation of tumor cells does not influenceviability of cells in the final product (data not shown).

DCs Pulsed with HHP-Frozen Tumor Cells Induce Tumor-Specific T Cells:

To investigate whether cryopreserved tumor cells expressing immunogeniccell death markers induce anti-tumor immunity, the ability of tumorcell-loaded DCs to activate tumor cell-specific T cell responses wasevaluated. Prostate (LNCap) and ovarian (SKOV3) tumor cells killed byHHP and HHP-frozen tumor cells were cocultured with immature DCs withsubsequent maturation with polyI:C. These DCs were then used asstimulators of autologous T cells, and the frequency of IFN-γ-producingT cells was analyzed 1 week later, after restimulation with tumorcell-loaded DCs. DCs pulsed with tumor cells killed by HHP induced agreater number of tumor-specific CD4⁺ and CD8⁺ IFN-γ-producing T cellscompared to immature DCs (FIG. 4A, B). Furthermore, DCs pulsed withtumor cells killed by HHP and cryopreserved induced significantly(*p<0.05) greater number of tumor-specific CD8⁺ IFN-γ-producing T cellsand non-significantly increased percentage of CD4⁺ T cells (FIG. 4A, B).

6.3 Discussion

The results show that the cryopreservation process does not abolish theimmunogenicity of HHP-treated tumor cells. In particular, the resultsdemonstrate that the cryopreserved HHP treated tumor cells express asignificantly higher amount of immunogenic markers compared tonon-frozen cells. The same level of expression of IMM markers HSP70 andHSP90 were detected after 2 weeks, 1, 2, and 3 months of cryo storage.The expression of CRT IMM marker was non-significantly decreased afterthe first month of storage. Nevertheless, the expression of IMM markersis still significantly higher compared to untreated cells during all thetested time.

The results also demonstrate that the cryopreservation of theHHP-treated tumor cells does not negatively influence the amount oftumor associated antigen. The expression of PSA, PSMA and Her2/Neuprotein on the surface of HHP and HHP-frozen cells was comparable.However, the complete cell lysate of HHP-treated tumor cells containedsignificantly higher amount of all three tested proteins detected bywestern blotting. This phenomena was even increased after thecryopreservation process in both prostate and ovarian tumor cells.

The results also demonstrate that the rate of phagocytosis of HHP-frozenprostate tumor cells was comparable to HHP-treated non-frozen tumorcells. In fact, more than 85% of pulsed dendritic cells with apoptoticbodies were detected. Furthermore, the level of expression ofmaturation-associated markers shows the similar activation status of DCspulsed with HHP-frozen tumor cells. Moreover, DCs pulsed withHHP-treated tumor cells efficiently stimulated tumor-specificIFN-γ-producing CD8⁺ and CD4⁺ T cells. However, the cryopreservationprocess of tumor cells significantly increased the percentage of CD8⁺IFN-γ-producing T cells.

The embodiments described herein are intended to be merely exemplary,and those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. All such equivalents areconsidered to be within the scope of the present invention and arecovered by the following claims.

All references (including patent applications, patents, andpublications) cited herein are incorporated herein by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

What is claimed is:
 1. A method for preparing a pharmaceuticalcomposition for use in immunotherapy comprising: a) inducing immunogeniccell death in a population of cancer cells; b) cryopreserving the cancercells undergoing immunogenic cell death in a cryopreservant; c) thawingthe cryopreserved cancer cells; and d) preparing a pharmaceuticalcomposition comprising the thawed cancer cells for use in immunotherapy.2. The method of claim 1, wherein the cancer cells are from a cancercell line.
 3. The method of claim 1, wherein the cancer cells originatefrom multiple cancer cell lines.
 4. The method of claim 1, wherein theimmunogenic cell death is induced by high hydrostatic pressure,anthracyclines, anti-EGFR antibodies, Big Potassium channel antagonists,bortezomib, cardiac glycosides, cyclophosphamide, GADD43/PP1 inhibitorsand mitomycin, irradiation by UV light or gamma rays, oxaliplatin,photodynamic therapy with hypericin, poly(I:C), or thapsigargin andcisplatin.
 5. The method of claim 1, wherein the cell death is inducedby high hydrostatic pressure.
 6. The method of claim 1 wherein the celldeath is induced by UV light.
 7. The method of claim 1, wherein theimmunogenic cell death is induced by anthracyclines.
 8. The method ofclaim 1, wherein the immunogenic cell death is induced by photodynamictherapy with hypericin.
 9. The method of claim 1, wherein thecryopreservant contains dimethyl sulphoxide at a concentration of atleast 5%.
 10. The method of claim 1, wherein the cryopreservant containsglycerol at a concentration of at least 5%.
 11. The method of claim 1,wherein the cells undergoing immunogenic cell death are cryopreserved ata temperature below −75° C.
 12. The method of claim 1, wherein thecryopreservation is performed by slow freezing.
 13. The method of claim1, wherein the cells undergoing immunogenic cell death are cryopreservedimmediately after induction of immunogenic cell death.
 14. The method ofclaim 1, wherein the cells undergoing immunogenic cell death arecryopreserved up to six hours after induction of immunogenic cell death.15. The method of claim 1, wherein the thawing is performed for between5 seconds and 10 minutes at a temperature between 32° C. and 40° C. 16.The method of claim 1, wherein the thawed cells are put back intoculture for a least 1 hour and not more than 6 hours before being usedfor the pharmaceutical preparation.
 17. The method of claim 1, whereinthe immunotherapy is for the treatment of cancer.
 18. The method ofclaim 17, wherein the immunotherapy comprises the treatment of a solidtumor cancer such as prostate cancer, ovarian cancer, lung cancer, renalcancer, colon cancer, breast cancer or glioblastoma.
 19. The method ofclaim 1 wherein the one or more of the hallmarks of immunogenic celldeath are preserved during cryopreservation.
 20. The method of claim 19,wherein the one or more of the hallmarks of cell death of are selectedfrom the group consisting of calreticulin, HSP70, and HSP90.
 21. Themethod of claim 1 wherein, after thawing, the cells undergoing celldeath retain one or more of the hallmarks of immunogenic cell death. 22.The method of claim 21, wherein the one or more of the hallmarks of celldeath of are selected from the group consisting of calreticulin, HSP70,and HSP90.
 23. A pharmaceutical composition for use in immunotherapyprepared by the method of claim
 1. 24. A kit comprising one or morecontainers comprising cryopreserved cancer cells undergoing immunogeniccell death.